WO2003035844A2 - Nouvelle histone methyltransferase et methodes d'utilisation - Google Patents

Nouvelle histone methyltransferase et methodes d'utilisation Download PDF

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WO2003035844A2
WO2003035844A2 PCT/US2002/034321 US0234321W WO03035844A2 WO 2003035844 A2 WO2003035844 A2 WO 2003035844A2 US 0234321 W US0234321 W US 0234321W WO 03035844 A2 WO03035844 A2 WO 03035844A2
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dim
cell
methylation
dna
compound
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WO2003035844A9 (fr
WO2003035844A3 (fr
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Eric U. Selker
Hisashi Tamaru
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The State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)

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  • FIELD This disclosure relates to histone methyltransferases, nucleic acids encoding such, and methods for the use of these molecules. It also relates to methods for influencing DNA methylation and gene activation, as well as systems and methods for identifying molecules that influence DNA methylation.
  • DNA methyltransferases also referred to as DNA methylases transfer methyl groups from the universal methyl donor S-adenosyl methionine to specific sites on a DNA molecule.
  • DNA methyltransferases transfer methyl groups from the universal methyl donor S-adenosyl methionine to specific sites on a DNA molecule.
  • Several biological functions have been attributed to the methylated bases in DNA. The most established biological function is the protection of the DNA from digestion by cognate restriction enzymes. The restriction modification phenomenon has been observed only in bacteria. Mammalian cells possess at least three methyltransferases; one of these (DNMT1) preferentially methylates cytosine residues on the DNA that are 5' (upstream) neighbors of guanine (forming the dinucleotide CpG).
  • HMTase histone methyltransferase
  • DIM-5 A novel histone methyltransferase (HMTase) has been identified in Neurospora, and is termed herein DIM-5. Nucleic acids encoding this enzyme, and the protein itself, are provided herein. Through characterization of DIM-5, it has been surprisingly discovered that the methylation of histones influences and controls the methylation of DNA presumably in regions proximal to the methylated histones. Thus, the systems provided herein illustrate for the first time a pathway involved in influencing and controlling DNA methylation and thereby controlling gene expression in eukaryotic cells.
  • This disclosure provides methods and compositions useful in regulating and influencing histone methylation, for instance methylation of the lysine 9 residue of histone H3, and particularly trimethylation in some embodiments, and thereby altering DNA methylation in eukaryotic cells. Also provided are methods for identifying molecules that interact with HMTases, for instance which inhibit or enhance the activity or histone-binding affinity or specificity of a HMTase, and therefore which are useful in influencing histone and/or DNA methylation in a target cell.
  • FIG. 1 Growth deficiencies of im-5 strains. Rates of apical growth often wildtype (dim ⁇ ) and ten mutant (dim-5) progeny of N2140 (dim-5, le ⁇ -2, pan-2, A) X N 185 (trp-4, ) were measured at 32 C using "race tubes" containing 1.5% sucrose Vogel's medium with pantothenate. The average growth rates and standard deviations of the wildtype and mutant strains were 4.9 H /-0.1 and 2.4 +/- 0.7 mm/hour, respectively. The growth rates of the ten wildtype strains were so similar that their plots are virtually superimposed.
  • FIG. 1 DNA methylation defect of dim-5 strains. Genomic DNA of wildtype (wt), a DNA methyltransferase mutant (dim-2) and the dim-5 mutant were digested with DpnW (D) or Sai ⁇ M (S) and analyzed by gel electrophoresis and Southern hybridization using probes for the indicated five methylated chromosomal regions. The DNAs were stained with ethidium bromide (total) to reveal the total digestion profiles generated with these isoschizomers. Blots were reprobed for unmethylated regions to confirm that digests were complete. The 9A20 region in the dim-5 strain shows an RFLP relative to the wild-type and dim-2 strains.
  • FIG 3A Map of genes revealed by BLASTx in leu-2/trp-4 interval of contig 1.1 18 (Assembly version 1. Neurospora Sequencing Project, Whitehead Institute/MIT Center for Genome Research, 2001 ). Regions of marked similarity (alignment scores >80) to genes in NCBI database are indicated (rectangles). A segment that was amplified to test two dim-5 candidates (hibD gene; blue; homologue of & pombe clr4; red with white intron) is shown expanded.
  • FIG 3B illustrates complementation of the dim-5 mutation
  • dim-5 strain N2145 was co-transformed with pB16 and 2.0kb Pst ⁇ -Xba ⁇ or 1.4 kb Ml ⁇ -Xba ⁇ fragment.
  • Genomic DNA from representative bml R transformants was analyzed by Southern hybridization for DNA methylation in the ⁇ 63 region using £coRI (E) and Ban ⁇ W (B). Methylation of the BamUl site (Margolin et al, Genetics 149: 1787- 1797, 1998) gives a 6.4 kb fragment, as illustrated. Results for representative transformants are shown with positive (wt) and negative (dim-5) controls.
  • FIG 4A Cartoon of methylated (hyg s ) or unmethylated (hyg R ) hph gene, flanked by methylation-inducing DNA segments that had been subjected to RIP (Irelan & Selker et al, Genetics 146:509-523, 1997).
  • FIG 4B Cartoon of methylated (hyg s ) or unmethylated (hyg R ) hph gene, flanked by methylation-inducing DNA segments that had been subjected to RIP (Irelan & Selker et al, Genetics 146:509-523, 1997).
  • FIG 4B Cartoon of methylated (hyg s ) or unmethylated (hyg R ) hph gene, flanked by methylation-inducing DNA segments that had been subjected to RIP (Irelan & Selker et al, Genetics 146:509-523, 1997).
  • FIG 4B Cartoon of methylated
  • FIG 4C Effect of dim-5 gene fragments on methylation at ⁇ 63.
  • the transformants illustrated in FIG 4B were analyzed for methylation as in FIG 3.
  • Figure 5A Amino acid alignment of conserved regions ofN. crassa DIM-5
  • FIG 5B Protein domain organization of DIM-5 and related proteins aligned at their C-termini with predicted number of amino acids and locations of Chromo, SET, and cysteine-rich (C-rich), domains indicated.
  • the N-terminal endpoints of recombinant proteins made in this study or previously (Rea et al, Nature 406:593-599, 2000) are indicated by vertical dashed lines.
  • FIG. 6 Histone methyltransferase activity of recombinant dim-5 protein.
  • Purified histones (20 ⁇ g; Boehringer Mannheim) were incubated for 6 hours at 20° C with or without purified GST-DIM-5 fusion protein (GST-DIM-5; ⁇ l ⁇ g) and 2.75 ⁇ Ci S-adenosyl-[methyl- H]-L- ethionine, as methyl donor.
  • Reaction products were fractionated by PAGE (16.5%), stained with Coomassie Blue (left) and then fluorographed (right) to detect methylation.
  • the positions of selected size standards, intact recombinant protein (*) and core histones are indicated.
  • FIG 7A Sequence of N-terminal segment of Neurospora histone H3 with residues presumed to be subject to methylation (m), acetylation (a) or phosphorylation (p) in red and residue implicated in silencing highlighted in yellow.
  • FIG 7B Sequence of N-terminal segment of Neurospora histone H3 with residues presumed to be subject to methylation (m), acetylation (a) or phosphorylation (p) in red and residue implicated in silencing highlighted in yellow.
  • FIG 7B Sequence of N-terminal segment of Neurospora histone H3 with residues presumed to be subject to methylation (m), acetylation (a) or phosphorylation (p) in red and residue implicated in silencing highlighted in yellow.
  • FIG 7B Sequence of N-terminal segment of Neurospora histone H3 with residues presumed to be subject to methylation (m), acetylation (a) or phosphorylation (p) in red and residue implicated
  • FIG 7C Southern analysis and sequencing of DNA from hyg R transformants. DNA of representative transformants (T) and a wildtype (wt) control grown non-selectively was analyzed with EcoK] and BamY ⁇ for methylation ( ) at ⁇ 63 as in FIG 3 and for ectopic alleles of hH3. Direct sequencing of hH3 PCR products confirmed the presence of both the wildtype and mutant alleles in representative strains (sequencing chromatograms).
  • FIG. 8 Structure-Based Sequence Alignment of SET Proteins.
  • the alignment includes (1 ) all known members of human SUV39 family: SUV39H1 (accession NP_003164), SUV39H2 (accession NP )78946), G9a (accession S30385), Eu-HMTl (accession AAM09024), SETDB 1
  • the residue number and secondary structural elements of DIM-5 are shown above the aligned sequences. Dashed lines indicate disordered regions. Specific regions include the N terminus (residues 25-62), the pre-SET (residues 63-146), the SET (residues 147-236 and 248-277), the signature motifs (SET residues 237-247 and 278 -285), and the post-SE I (residues 299-308).
  • the amino acids highlighted are invariant (white against black) and conserved (white against gray) among almost all members of the SUV39 family. The number in parentheses indicates the number of amino acids inserted relative to the alignment.
  • FIG 9A Front view of ribbons diagram (Carson, 1997) (top, stereo; bottom, mono). The protein is shaded according to the regions indicated in FIG 8, and the three zinc ions are shown as balls (as in FIG 9C).
  • FIG 9B Side view.
  • a dashed line indicates the disordered amino acids between strand ⁇ l 7 (magenta) and the post-SET segment.
  • FIG 9C Stereo diagram of the triangular zinc cluster. Three zinc ions are shown as three numbered balls, the bridging (B) and nonbridging (NB) cysteine residues are indicated. The pre-SET sequence of DIM-5 is shown above. Both Cys-rich segments coordinate the one (red) and two (blue) zinc ions jointly, while the three (green) zinc ion is coordinated solely by the five-Cys segment.
  • Figure 10 Enzymatic Properties of Recombinant DIM-5. This figure shows HKMT activity as functions of (FIG 10A) temperature, (FIG 10B) salt concentration, (FIG 10C) pH, and
  • FIG I 0D AdoMet crosslinking as a function of pH.
  • the buffers used were 50 mM Na citrate for pH 5.0-6.0, MES for pH 6.0-6.5, HEPES for pH 7.0- 7.5, Tris for pH 8.0- 8.5, Bicine for pi I 9.0, and glycine for pH 9.35-10.7.
  • Na citrate and Mes are used for pH 6.0.
  • FIG 10E shows relative activities of DIM-5 mutants with conservative point mutations. All mutant proteins were expressed to level similar to that of the wild-type, though some were less soluble, and all were monomeric, suggesting that none of the mutations caused gross aggregation of the protein.
  • mutant enzymes were used, the activities were compared to that of serial dilutions of wild-type enzymes purified in the same way, and the specific activity of mutant proteins relative to wild-type was estimated.
  • the activities shown are averages of at least two measurements.
  • FIG 10F shows fluorographic results of an AdoMet crosslinking experiment at pH 8.0, along with results of Coomassie staining to control for the amount of mutant protein tested in FIG 10E.
  • FIG 11 The Cofactor Binding and Active Site in DIM-5. Close-up view of the proposed cofactor binding site and the adjacent active site (top, stereo; bottom, mono). The difference electron density map (grey hatch structure) is contoured at 5.5 ⁇ ; the water molecules are numbered 1-4. Dashed lines indicate the hydrogen bonds.
  • the water at site 2 is hydrogen bonded to the main chain carbonyl oxygen atom of R238 and to the water molecules at sites 1 and 3, which in turn interacts with the side chain carbonyl oxygen of N241 and the side chain hydroxyl oxygen of Y204, respectively.
  • FIG 12A is a front view of GRASP surface (Nicholls e/ ot, Proteins 1 1 :281-296, 1991).
  • the difference electron density map (black) is contoured at 5.5 ⁇ .
  • Strand BI O includes L205, F206 and A207; N241 , H242, and Y283 are shown below, and C244 includes Q5, T6, A7, R8, K9, and S10, each of which is shaded.
  • FIG 12B is a superimposition image of Drosophila HPI ⁇ strand (Jacobs and Khorasanizadeh, Science 295:2080-2083, 2002; PDBcode 1 KNA) and DIM-5 strand B10. Dashed lines indicate the hydrogen bonds between HP] and H3 peptide.
  • the DIM-5 residues on the other side of the HPI peptide are Y283, V284, and N285.
  • the dimethylated (methyl groups in black) target nitrogen atom occupies water site 2 (see FIG 1 1).
  • the sequence of histone H3 peptide is shown at the bottom; both K4 and 14 are five residues away from K9.
  • FIG 12C shows the docked H3 peptide lying in the putative peptide binding cleft.
  • the cleft extends in both directions following turns as indicated.
  • FIG 12D is a superimposition of active site NPPY residues of Taq ⁇ DNA-adenine amino MTase (Goedecke et al, Nat. Struct. Biol. 8: 121-125, 2001 ; PDB code 1G38) and the proposed DIM-5 active site residues N241 , H242, and Y283.
  • the Tyr in both cases is hydrogen bonded to a main chain amide nitrogen atom (dashed bonds).
  • Figure 13 Metal Chelators Inhibit DIM-5 Activity.
  • FIG 13A shows analysis of zinc content of DIM-5 with and without EDTA treatment. DIM-5 protein was incubated with 20 mM EDTA for two days, at which time HKMT activity was no longer detectable.
  • FIG 13B is a bar graph showing relative activity.
  • Purified DIM-5 protein (1 mg/ml in 20 mM glycine [pH 9.8], 5% glycerol) was incubated with various concentration of 1 , 10-phenanthroline or EDTA for 18 hours at 4°C. The enzyme was diluted 80-fold and assayed for HKMT activity under standard conditions, except that no DTT was present.
  • FIG 13C shows fluorographic results of AdoMet crosslinking in the presence of the indicated levels of EDTA.
  • Figure 14 DIM-5 trimethylates lysine 9 of histone H3 efficiently in vitro.
  • FIG 14A illustrates DIM-5 activity with histone H3 peptide unmodified, dimethylated, or trimethylated at Lys9.
  • 0.5 ⁇ g unmodified, di- or trimethyl-Lys9 histone H3 peptide (ARTKQTARKSTGGKA; positions 1-15) was incubated for one hour at 16 °C with 0.5 ⁇ g purified recombinant DIM-5 protein (Zhang e/ ⁇ /., Ce/ 1 1 1 : 1 17-127, 2002) and 1.1 ⁇ Ci S-adenosyl-[methyl- 3H]-L-methionine ( 3 H SAM).
  • Reaction products were fractionated by SDS-PAGE ( 16.5%), fixed with 10% gluthalaldehyde for 15 minutes and fluorographed to detect methylation as described (Tamaru & Selker, Nature 414:277-283, 2001). Each peptide was assayed independently twice (1 & 2).
  • FIG 14B and 14C illustrate the determination of amino acid position of H3 peptides methylated by DIM-5.
  • DIM-5 reactions were carried out as in panel A with either unmodified (FIG 14B) or dimethyl-Lys9 (FIG 14C) H3 peptides (ARTKQTARKSTGGKAPRKQL; positions 1-20).
  • Reaction products were subject to amino terminal sequencing and incorporation of labeled methyl groups into individual amino acid residues was detected by scintillation counting of each a ino acid fraction.
  • the amino acid sequence is shown below and lysine (K) residues are numbered. Fractions containing free 3 H SAM are indicated in gray.
  • FIG 14D through FIG 14G show mass spectrometry analyses of DIM-5 products from unmodified or dimethyl-Lys9 H3 substrates. Reactions were initiated by addition of 100 ⁇ M unmodified (FIG 14D) or dimethyl-Lys9 (FIG 14E) H3 substrate (TKQTARKSTGGKA; positions 3- 15) to a 20 ⁇ l mixture of 50 M Glycine (pH 9.8), 10 mM DTT, 750 ⁇ M S-adenosyl-L-methionine and 2 ⁇ g DIM-5. After incubation at room temperature for the indicated times, reactions were stopped by addition of TFA to 0.5%.
  • FIG 15A illustrates the specificity of antibodies for methylated Lys9 of histone H3.
  • samples from a 2x dilution series were spotted onto a nitrocellulose membrane, stained with Ponceau S (bottom) and analyzed by immunoblotting (top) using anti-H3 dimethyl-Lys9 (Nakayama et al, Science 292: 1 10- 1 13, 2001) or anti-H3 trimefhyl-Lys9 (Cowell et al, Chromosoma 1 1 1 :22-36, 2002) antibodies.
  • FIG 15B shows Southern analyses of unmethylated (pen and hH4) and methylated (?/ and punt) chromosomal regions of Neurospora crassa.
  • FIG 15C is a diagram of endogenous Vietnamese ura4 allele carrying deletion (ura4DS/E) and an ectopic heterochromatic ura4 allele integrated in cenl (cenl::ura4) in Schizosaccharomyces pombe strain SPG 1355.
  • PCR with primers ura4DS/E# ⁇ and ura4DS/E#2 (Nakayama et al, Cell 101 :307-317, 2000) (indicated by arrows) generates products of distinctive lengths from ura4DS/E and cenl::ura4.
  • the central component (cntl) and part of the inverted repeats (imrlR and otrlR) of cenl are also represented.
  • ChIP with TV. crassa and S. pombe extracts was carried out as described herein.
  • Mixtures of extracts of N. crassa wild-type strain 740R23-IVA and S. pombe strain SPG 1355 were incubated with anti-H3 dimethyl-Lys4 (Upstate Biotechnology), anti-H3 dimethyl-Lys9 (Upstate Biotechnology) or anti-H3 trimethyl-Lys9 (Cowell et al, Chromosoma 1 1 1 :22-36, 2002) antibodies, or incubated without antibody (no antibody control).
  • FIG. 17 DIM-5 is responsible for histone H3 Lys9 trimethylation associated with methylated DNA.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NO: 1 shows a dim-5 encoding sequence. This sequence is believed to include an intron from position 49 through 172, which when spliced out produces DIM-5' (SEQ ID NO: 2).
  • An alternative splicing variant is also possible, wherein the intron is from position 49 through 199; when spliced out, this produces DIM-5" (SEQ ID NO: 4).
  • SEQ ID NO: 2 shows the nucleic acid sequence of DIM-5', one of two splice variants, and the encoded amino acid sequence.
  • SEQ ID NO: 3 shows the deduced amino acid sequence of one variant of DIM-5, encoded by the DIM-5' cD A.
  • SEQ ID NO: 4 shows the nucleic acid sequence of DIM-5", one of two splice variants, and the encoded amino acid sequence.
  • SEQ ID NO: 5 shows the deduced amino acid sequence of one variant of DIM-5, encoded by the DIM-5" cDNA.
  • SEQ ID NOs: 6-21 show DNA primer pairs used for PCR in vitro amplification reactions as described herein.
  • these primers were used to amplify region 1 D21 (SEQ ID NOs: 6 and 7); region 9a20 (SEQ ID NOs: 8 and 9); region hibDI dim-5 (SEQ ID NOs: 10 and 1 1); region dim- 5 ORF (SEQ ID NOs: 12 and 13); region 5 '-GST-DIM-5 (SEQ ID NOs: 14 and 15); region H3L9 (SEQ ID NOs: 16 and 17); region H3R9 (SEQ ID NOs: 18 and 19); and region H3-ORF (SEQ ID NOs: 20 and 21).
  • SEQ ID NOs: 22-29 show DNA primers used for PCR in vitro amplification reactions of ⁇ (SEQ ID NOs: 22 and 23), punt (SEQ ID NOs: 24 and 25), pen (SEQ ID NOs: 26 and 27), and hH4 (SEQ ID NOs: 28 and 29) as described in Example 12.
  • DMTase DNA methyltransferase
  • HMTase histone methyltransferase
  • Alcohol refers to a chemical compound with the structure R-OH, wherein R is alkyl, especially lower alkyl (for example in methyl, ethyl, or propyl alcohol).
  • R is alkyl, especially lower alkyl (for example in methyl, ethyl, or propyl alcohol).
  • An alcohol may be either linear or branched, such as isopropyl alcohol.
  • Alkyl refers to a cyclic, branched, or straight chain alkyl group containing only carbon and hydrogen, and unless otherwise mentioned contains one to twelve carbon atoms. This term is further exemplified by groups such as methyl, ethyl, n-propyl, isobutyl, t-butyl, pentyl, pivalyl, heptyl, adamantyl, and cyclopentyl. Alkyl groups can either be unsubstituted or substituted with one or more substituents, e.g.
  • alkyl refers to a cyclic, branched or straight chain monovalent alkyl radical of one to five carbon atoms.
  • alkyl groups can also be unsubstituted or substituted, where a specific example of a substituted alkyl is 1 , 1 -dimethyl propyl.
  • Alkoxy refers to a substituted or unsubstituted alkoxy, where an alkoxy has the structure -O-R, where R is substituted or unsubstituted alkyl.
  • R is an unsubstituted alkyl.
  • substituted alkoxy refers to a group having the structure -O- R, where R is alkyl which is substituted with a non-interfering substituent.
  • amino refers to a chemical functionality -NR,R 2 where R
  • Analog, derivative or mimetic An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington (The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28).
  • a derivative is a biologically active molecule derived from the base structure.
  • a mimetic is a biomolecule that mimics the activity of another biologically active molecule.
  • Biologically active molecules can include chemical structures that mimic the biological activities of a compound.
  • Animal Living multi-cellular organisms, for instance a vertebrate (a category that includes, for example, mammals, and birds). The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.
  • Anti-proliferative activity An activity of a molecule, e g , a compound, which reduces proliferation of at least one cell type, but which may reduce the proliferation (either in absolute terms or in rate terms) of multiple different cell types (e g , different cell lines, different species, etc.). In specific embodiments, an anti-proliferative activity will be apparent against cells (either in vitro or in vivo) that exhibit a hyper-proliferative condition, such as is characteristic of certain disorders or diseases.
  • an anti-proliferative activity can be an anti-tumor or anti-neoplastic activity of a compound.
  • Such molecules will be useful to inhibit or prevent or reduce cellular proliferation or growth, e g., in a tumor, such as a malignant neoplasm.
  • aryl refers to a monovalent unsaturated aromatic carbocyclic group having a single ring (e g phenyl) or multiple condensed rings (e g naphthyl or anthryl), which are optionally unsubstituted or substituted with, e g , halogen, alkyl, alkoxy, mercapto (-SH), alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1 -yl, piperazin-1 -yl, or other functionality.
  • Carboxyl This term refers to the radical -COOH, and substituted carboxyl refers to -COR where R is alkyl, lower alkyl or a carboxylic acid or ester.
  • DNA deoxyribonucleic acid
  • DNA is a long chain polymer which comprises the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid (RNA)).
  • the repeating units in DNA polymers are four different nucleotides, each of which comprises one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached.
  • Triplets of nucleotides (referred to as codons) code for each amino acid in a polypeptide, or for a stop signal.
  • codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.
  • any reference to a DNA molecule is intended to include the reverse complement of that DNA molecule. Except where single-strandedness is required by the text herein, DNA molecules, though written to depict only a single strand, encompass both strands of a double-stranded DNA molecule.
  • Halogen refers to fluoro, bromo, chloro, and iodo substituents.
  • Heterocycle refers to a monovalent saturated, unsaturated, or aromatic carbocyclic group having a single ring (e g benzyl, morpholino, pyridyl or furyl) or multiple condensed rings (e g naphthyl, quinolinyl, indolizinyl or benzo[b]thienyl) and having at least one heteroatom, defined as N, O, P, or S, within the ring, which can optionally be unsubstituted or substituted with, e g halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1 -yl, piperazin-1-yl, or other functionality.
  • a single ring e
  • Histone methyltransferase is defined as an enzyme that adds one or more methyl groups to one or more positions of a histone.
  • DIM-5 is a representative example of a HMTase; it can add one or more methyl groups to lysine 9 of histone H3 or to fragments thereof.
  • Hydroxyl This term refers to the chemical group -OH.
  • Hyper-proliferative disorder A disorder characterized by abnormal proliferation of cells, and generically includes skin disorders such as psoriasis as well as benign and malignant tumors of all organ systems.
  • a pharmaceutically acceptable fluid composition comprising at least one active ingredient, e.g., a compound that binds to and or inhibits a HMTase.
  • the active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally comprise minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, and pH buffering agents and the like.
  • non-toxic auxiliary substances such as emulsifying agents, preservatives, and pH buffering agents and the like.
  • In vitro amplification Techniques that increases the number of copies of a nucleic acid molecule in a sample or specimen.
  • An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to nucleic acid template in the sample.
  • the primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid.
  • the product of in vitro amplification may be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing, using standard techniques.
  • in vitro amplification techniques include strand displacement amplification (see U.S. Patent No. 5,744,31 1); transcription-free isothermal amplification (see U.S. Patent No. 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP-A-320 308); gap filling ligase chain reaction amplification (see U.S. Patent No.
  • Methylation A chemical or biochemical process of introducing a methyl group into an organic molecule.
  • DNA methylation the addition of a methyl group onto a nucleotide, is a postreplicative covalent modification of DNA that is catalyzed by the DNA methyltransferase enzyme (DMeTase) (Koomar et al, Nucl. Acids Res. 22:1-10, 1994; and Bestor et al, J. Mol. Biol.
  • Proteins also can be methylated, as described herein for histone methylation.
  • DNA methylation can serve as a mechanism for changing the structure of DNA without altering its coding function or its sequence.
  • DNA methylation is a heritable, reversible and epigenetic change. It can alter gene expression, particularly by suppressing or inactivating genes, which has profound developmental and disease consequences. Methylation of CpG islands that are associated with tumor suppressor genes can cause decreased gene expression. Increased methylation of such regions often leads to reduction of normal gene expression, which may cause the selection of a population of cells having a selective growth advantage and thus are or become malignant.
  • DNA hypermethylation refers to an increased or high level
  • nucleic acid molecule e.g., a CpG island
  • a promoter region e.g., a promoter region
  • DNA hypomethylation refers to a decreased or low level (below a reference level, such as wild-type or other basal level) of DNA methylation at a specific site on a nucleic acid molecule (e.g., a CpG island), or more generally in a genome or region of a genome (e.g., a promoter region).
  • a nucleic acid molecule e.g., a CpG island
  • a promoter region e.g., a promoter region
  • DNA hypomethylating agent refers to an agent that reduces or reverses DNA methylation, either at a specific site (e.g., a specific CpG island) or generally throughout a genome.
  • Hypomethylating agents can be referred to as possessing "hypomethylating activity.”
  • such activity is measured by determining the methylation state and/or level of a specific DNA molecule or site therein, or the general methylation state of a cell, on parallel samples that have and have not been treated with the hypomethylating agent (or putative hypomethylation agent). A reduction in methylation in the treated (versus the untreated) sample indicates that the agent has hypomethylating activity.
  • the methylation level of a target biological molecule is reduced by at least 5% upon treatment with a hypomethylating agent; in other embodiments it is reduced by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, or by at least 50% compared to an untreated sample.
  • Methylation-mediated or -related condition/disease/disorder A biological condition, disease or disorder of a subject that is associated with, caused by, or influenced by the methylation state (e.g., the extent of methylation) of a DNA sequence, the level of methylation throughout the genome of the subject, and/or the level of methylation of a protein or residue within a protein or proteins.
  • methylation state e.g., the extent of methylation
  • Some hypermethylation-associated diseases, disorders, and conditions are characterized by exhibiting hypermethylation of one or more target biological molecules.
  • Such diseases, disorders, and conditions therefore can be identified by examining the methylation state (or level) of target molecules in a subject known to or suspected of suffering therefrom; a high level of specific or general methylation indicates that the disease/disorder/condition is hypermethylation-associated. It is beneficial to treat (or prevent) such diseases, disorders, and conditions with HMTase-activity altering compositions, for instance compositions identified using the methods described herein.
  • hypomethylation-associated diseases, disorders, and conditions are characterized by exhibiting hypomethylation of one or more target biological molecules.
  • hypomethylation-associated diseases/disorders/conditions can be identified by examining the methylation state (or level) of target molecules in the subject known to or suspected of suffering therefrom.
  • Nucleoside includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine, or synthetic analogs thereof.
  • Nucleotide is a nucleoside plus a phosphate, and forms one monomer in a polynucleotide.
  • a nucleotide sequence refers to the sequence of bases in a polynucleotide.
  • Oligonucleotide is a plurality of joined nucleotides joined by native phosphodiester bonds, between about 6 and about 300 nucleotides in length.
  • An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions.
  • oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide.
  • Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA.
  • Particular oligonucleotides and oligonucleotide analogs can include linear sequences up to about 200 nucleotides in length, for example a sequence (such as DNA or RNA) that is at least 6 bases, for example at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long, or from about 6 to about 50 bases, for example about 10-25 bases, such as 12, 15 or 20 bases.
  • a sequence such as DNA or RNA
  • Ortholog Two nucleic acid or amino acid sequences are orthologs of each other if they share a common ancestral sequence and diverged when a species carrying that ancestral sequence split into two species. Orthologous sequences are also homologous sequences.
  • parenteral Administered outside of the intestine, e.g., not via the alimentary tract.
  • parenteral formulations are those that will be administered through any possible mode except ingestion. This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, intraperitoneally, or subcutaneously, and various surface applications including intranasal, intradermal, and topical application, for instance.
  • Pharmaceutical agent or drug A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.
  • compositions and formulations suitable for pharmaceutical delivery are conventional. See, for instance, Remingto 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), which describes compositions and formulations suitable for pharmaceutical delivery.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions e.g., powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Pre-cancerous lesion This term includes syndromes represented by abnormal neoplastic, including dysplastic, tissue changes. Examples include dysplastic growths in colonic, breast, prostate, or lung tissues, or conditions such as dysplastic nevus syndrome (a precursor to malignant melanoma of the skin), polyposis syndromes, colonic polyps, precancerous lesions of the cervix (such as cervical dysplasia), esophagus, lung, prostatic dysplasia, prostatic intraneoplasia, breast and/or skin and related conditions (e.g., actinic keraosis), whether the lesions are clinically identifiable or not.
  • Prodrug Any molecule that undergoes in vivo metabolic conversion to one or more pharmacologically active compound(s).
  • Tumor A neoplasm that may be either malignant or non-malignant.
  • Tumors of the same tissue type refers to primary tumors originating in a particular organ (such as breast, prostate, bladder, or lung). Tumors of the same tissue type may be divided into tumor of different sub-types (a classic example being bronchogenic carcinomas (lung tumors) which can be an adenocarcinoma, small cell, squamous cell, or large cell tumor).
  • a compound with the potential for treating a methylation-related disease or condition such as neoplasia
  • Such methods include determining a histone methyltransferase (HMTase) inhibitory activity of the compound, wherein high HMTase inhibition activity identifies that the compound has potential for treating a methylation-related disease or condition.
  • HMTase inhibitory activity includes a histone H3 methyltransferase activity.
  • Some specific examples of the provided methods further include determining a DIM-5 inhibitory activity of the compound, wherein high DIM-5 inhibitory activity identifies that the compound has potential for treating a methylation-related disease or condition.
  • Still other examples of the methods include determining whether the compound inhibits tumor cell growth in a culture, wherein inhibition of tumor cell growth further identifies that the compound has potential for treating a methylation-related disease or condition, and/or determining whether the compound inhibits or reverses DNA methylation in a cell, wherein inhibition or reversal of DNA methylation in the cell further identifies that the compound has potential for treating a methylation-related disease or condition, and/or determining whether the compound induces apoptosis of a tumor cell, wherein induction of apoptosis further identifies that the compound has potential for treating a methylation-related disease or condition.
  • kits for treating a methylation-related disease or condition also include determining whether the compound being tested inhibits tumor cell growth (e.g., the growth of a mammalian tumor) in a sample (either in vivo or in vitro), wherein inhibition of tumor cell growth further identifies that the compound is useful for treating a methylation-related disease or condition.
  • tumor cell growth e.g., the growth of a mammalian tumor
  • a sample either in vivo or in vitro
  • Still further embodiments provided herein include methods of selecting a compound for inhibition of a methylation-related disease or condition, which method involves determining neoplastic cell growth inhibitory activity of the compound; determining HMTase inhibitory activity; and selecting a compound that exhibits neoplastic cell growth inhibitory activity and high HMTase inhibition activity as a compound to inhibit the methylation-related disease or condition.
  • the methylation-related disease or condition involves disregulated cell growth, mo ⁇ hology, or division, and for instance in some instances involves a methylation-related disease or condition (e.g. a neoplasia or a neoplastic growth type).
  • Still further examples of methods provided herein further involve determining whether the test compound induces apoptosis in a cell; and selecting compounds that induce apoptosis for use and/or further testing.
  • Also provided herein are methods for identifying compounds for treatment of a methylation- related disease or condition which methods involve determining HMTase inhibitory activity of the compounds; and identifying those compounds for treating a methylation-related disease or condition if the compounds exhibit high HMTase inhibition activity.
  • methods of reducing, preventing or reversing DNA methylation in a cell which methods involve administering a hypomethylating effective amount of a HMTase inhibitory compound to the cell (e.g., a bacterial cell, a protist cell, a fungal cell, a plant cell, or an animal cell), thereby reducing, preventing or reversing DNA methylation in the cell.
  • a nucleic acid in the cell is known to be or suspected of being hypermethylated.
  • the cell is a hyper-proliferative cell (e.g., a mammalian tumor cell).
  • This disclosure further provides methods of treating or ameliorating a hypermethylation- related disease, condition, or disorder (e.g., a hyper-proliferative disease) in a subject, which methods involve administering to the subject a hypomethylating effective amount of a HMTase inhibitory compound, which compound is optionally administered in the form of a pharmaceutical composition.
  • a hypermethylation- related disease, condition, or disorder e.g., a hyper-proliferative disease
  • Another provided embodiment is a method of ameliorating a tumorigenic state of a cell, comprising administering a hypomethylating effective amount of a HMTase inhibitory compound (optionally administered in the form of a pharmaceutical composition) to the cell to reduce methylation of cytosine in a CpG dinucleotide in the cell, thereby ameliorating the tumorigenic state of the cell, in specific examples of this method, the method further involves administering an anticancer agent to the cell.
  • kits which kits may optionally include instructions for carrying out a method with one or more components of the kit.
  • kits include kits for inhibiting a DNA methyltransferase, which comprise an amount of a HMTase inhibitory compound effective to inhibit methylation of at least one DNA target.
  • included instructions include directions for administering at least one dose of the therapeutic substance to the subject in need of such treatment, for instance a methylation-related disease or condition ameliorating substance administered to a patient known or suspected of suffering from a methylation-related disease or condition.
  • compositions provided in the kits disclosed herein optionally can be provided in the form of a pharmaceutical composition.
  • Additional embodiments are purified proteins, which proteins have an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 5, or conservative substitutions thereof.
  • these proteins are functional DNA methyltransferases, one of which is DIM-5.
  • nucleic acid molecules encoding such proteins e.g., the nucleotide sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4
  • recombinant nucleic acid molecules that include a promoter sequence operably linked to such a nucleic acid molecule, and transgenic cells containing one of these recombinant nucleic acid molecules.
  • DNA methylation is involved in epigenetic processes such as X-inactivation, imprinting and silencing of transposons. It has been demonstrated previously that dim-2 encodes a DNA methyltransferase responsible for all known cytosine methylation in Neurospora crassa. Here we describe and disclose that another Neurospora gene, dim-5, is required for DNA methylation as well as for normal growth and full fertility. We mapped dim-5 and identified it by transformation with a candidate gene. The mutant has a nonsense mutation in a SET domain of a gene related to histone methyltransferases involved in heterochromatin formation in other organisms.
  • Cytosine methylation is essential for normal development of mammals and plants. Mutations in any of the three known DNA methyltransferase (DMTase) genes of the mouse (Dnmtl, Dnmt3a and Dnmt3b) are lethal, either during embryogenesis or soon thereafter (Li et al, Cell 69:915-926, 1992; Okano et al, Cell 99:247-257, 1999). In humans, a syndrome characterized by immunodeficiency, centromere instability, and facial anomalies, results from mutations in the DNMT3B gene (Xu, Nature 402 : 187- 191 , 1999).
  • DMTase DNA methyltransferase
  • DNA methylation is not essential in the filamentous fungus Neurospora crassa, facilitating investigations of DNA methylation in this organism.
  • a step to explore the control and mechanism of cytosine methylation, which remain largely unknown in eukaryotes we searched for methylation mutants in Neurospora.
  • a screen of strains surviving a chemical mutagenesis yielded one mutant completely defective in methylation (dim-2) and another with an approximately 50% reduction in total DNA methylation (dim-3) (Foss et al, Science 262:1737-1741, 1993).
  • the dim-2 gene has recently been isolated and demonstrated to encode a DMTase responsible for both de novo and maintenance methylation at both symmetrical and non-symmetrical sites (Kouzminova & Selker, EMBO Journal 20:4309-4323, 2001). Mutations in dim-2 relieve silencing of methylated genes (Rountree & Selker, Genes Dev. 11 :2383-2395, 1997; Cambareri et al, Genetics 143: 137-146, 1996), but do not noticeably affect growth or development (Kouzminova & Selker, EMBO Journal 20:4309-4323, 2001).
  • Transformation experiments confirmed that the candidate gene is dim-5 and biochemical tests on recombinant DIM-5 demonstrated that this protein methylates histone H3.
  • histone methylation controls DNA methylation was supported by demonstrating that replacements of lysine 9 in histone H3 cause loss of DNA methylation in vivo. This is explained in additional detail in the Examples, below.
  • heterochromatic state of the pericentric heterochromatin in mammals (Rea et al, Nature 406:593-9, 2000; Melcher et at, Mol Cell Biol 20:3728-41 , 2000; Peters et al, Cell 107:323-37, 2001), the silent mating type region and centromeres in Schizosaccharomyces pombe (Nakayama et al, Science 292: 110-3, 2001 ; Noma et al, Science 293:1150-5, 2001), the inactive X chromosome (Peters et al, Nat Genet 30:77-80, 2001 ; Heard et al, Cell 107:727-38, 2001), and at least some DNA methylation in Arabidopsis thaliana (Jackson et al, Nature 416:556-60, 2002; Johnson et al, Curr Biol 12:1360, 2002) depends on methylation of histone H3 lysine 9.
  • histone methyltransferases e.g., DIM-5, Clr-4 and Su(var)3-9
  • modifications to the N-terminal tail of the target histone such as (but not limited to) acetylation, methylation, and phosphorylation of particular residues (e.g., lysines 4, 9, and 14, serine 10 and probably other sites such as lysines 18, 23, 27, and 36).
  • H3 histone methylases function at least in part to "integrate" the information provided in the form of modifications to H3 and quite possibly similar information on other molecules (e.g., histone H4, histone H2A and histone H2B).
  • DNA methylation is controlled, at least in part, by histone methylation
  • DNA methylation should be affected by a variety of signals (e.g., other histone modifications) that influence H3 histone methyltransferases. Based on the work described herein, it is now apparent that procedures and drugs that influence (inhibit or stimulate) these underlying modifications also could be useful to influence DNA methylation, and therefore could be used (among other things) to clinically to treat conditions associated with hyper- or hypo-methylation of DNA.
  • RNA is extracted from cells by any one of a variety of methods well known to those of ordinary skill in the art.
  • Sambrook et al. In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989
  • Ausubel et al. In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998) provide descriptions of methods for RNA isolation. Any cell line derived from a non-DIM-5 deleted subject would be suitable.
  • RNA is then used as a template for performing the reverse transcription- polymerase chain reaction (RT-PCR) amplification of cDNA.
  • RT-PCR reverse transcription- polymerase chain reaction
  • Methods and conditions for RT-PCR are described in Kawasaki et al, In PCR Protocols, A Guide to Methods and Applications, Innis et al. (eds.), 21-27, Academic Press, Inc., San Diego, California, 1990.
  • the selection of PCR primers will be made according to the portions of the cDNA which are to be amplified. Primers may be chosen to amplify small segments of a cDNA or the entire cD A molecule.
  • Variations in amplification conditions may be required to accommodate primers and amplicons of differing lengths and composition; such considerations are well known in the art and are discussed in Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990).
  • primers and amplicons may be derived from the provided DIM-5-encoding sequence in order to amplify particular regions of the molecule.
  • Oligonucleotides derived from the provided DIM-5 sequences provided may be used in such sequencing methods.
  • Orthologs of DIM-5 can be cloned in a similar manner, where the starting material consists of cells taken from a non-human species. Orthologs will generally share at least 50% sequence homology with one or more of the disclosed DIM-5 encoding sequences. Where the species is more closely related to Neurospora, the sequence homology will in general be greater. Closely related orthologous DIM-5 molecules may share at least 75%, at least 80%, at least 90%, at least 95%, or at least 98% sequence homology with the disclosed sequences (e.g., SEQ ID NO: 1, 2, and/or 4).
  • Oligonucleotides derived from the DIM-5 encoding sequences are encompassed within the scope of the present invention.
  • Oligonucleotide primers may comprise a sequence of at least 10 consecutive nucleotides of the DIM-5 nucleic acid sequence. To enhance amplification specificity, oligonucleotide primers comprising at least 15, 25, 30, 35, 40, 45, 50, or 100 or more consecutive nucleotides of these sequences may also be used. These primers for instance may be obtained from any region of the disclosed sequences.
  • the DIM- 5 cD A, ORF and gene sequences may be apportioned into about halves or quarters based on sequence length, and the isolated nucleic acid molecules (e.g., oligonucleotides) may be derived from the first or second halves of the molecules, or any of the four quarters.
  • the DIM-5 cD A, shown in SEQ ID NO: 1, can be used to illustrate this.
  • the portion of a prototypical DIM-5 encoding sequence shown in SEQ ID NO: 1 is 1081 nucleotides in length and so may be hypothetical ly divided into about halves (nucleotides 1-540 and 541-1081) or about quarters (nucleotides 1-270, 271-540, 541- 81 1 and 812-1081).
  • Nucleic acid molecules may be selected that comprise at least 10, 15, 20, 25, 30, 35, 40, 50, or 100 or more consecutive nucleotides of any of these or other portions of a DIM-5 encoding sequence, or of the 5' or 3' flanking regions.
  • Variant DIM-5 proteins include proteins that differ in amino acid sequence from the DIM-5 sequences disclosed but that share at least 50% amino acid sequence homology with the provided DIM-5 protein. Other variants will share at least 60%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 98% amino acid sequence homology.
  • Manipulation of the nucleotide sequence of DIM-5 using standard procedures, including for instance site-directed mutagenesis or PCR can be used to produce such variants.
  • the simplest modifications involve the substitution of one or more amino acids for amino acids having similar biochemical properties. These so-called conservative substitutions are likely to have minimal impact on the activity of the resultant protein, so long as they do not affect amino acids in any active sites and/or binding pockets.
  • Table 1 shows amino acids that may be substituted for an original amino acid in a protein, and which are regarded as conservative substitutions.
  • Val ile; leu More substantial changes in enzymatic function or other protein features may be obtained by selecting amino acid substitutions that are less conservative than those listed in Table 1 Such changes include changing residues that differ more significantly in their effect on maintaining polypeptide backbone structure (e'g , sheet or helical conformation) near the substitution, charge, or hydrophobicity of the molecule at the target site, or bulk of a specific side chain
  • substitutions are generally expected to produce the greatest changes in protein properties (a) a hydrophilic residue (e g , seryl or threonyl) is substituted for (or by) a hydrophobic residue (e g , leucyl, isoleucyl, phenylalanyl, valyl or alanyl), (b) a cysteine or proline is substituted for (or by) any other residue, (c) a residue having an electropositive side chain (e g , lysyl, arginyl, or histadyl) is substitute
  • Variant DIM-5-encod ⁇ ng sequences may be produced by standard DNA mutagenesis techniques, for example, M13 primer mutagenesis Details of these techniques are provided in Sambrook et al (In Molecular Cloning A Laboratory Manual, CSHL, New York, 1989), Ch 15
  • variants may be created which differ in minor ways from the DIM-5 sequences disclosed DNA molecules and nucleotide sequences that are derivatives of those specifically disclosed herein, and which differ from those disclosed by the deletion, addition, or substitution of nucleotides while still encoding a protein that has at least 70% sequence identity with the DIM-5 sequence disclosed (SEQ ID NO 1 , 2, and/or 4), are comprehended by this mvention
  • nucleic acid molecules that share at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% or more nucleotide sequence homology with the disclosed DIM-5 sequences
  • variants may differ from the disclosed sequences by alteration of the coding region to fit the codon usage bias of the particular organism into which the molecule is to be introduced
  • the coding region may be altered by taking advantage of the degeneracy of the genetic code to alter the coding sequence such that, while the nucleotide sequence is substantially altered, it nevertheless encodes a protein having an amino acid sequence substantially similar to the disclosed DIM-5 protein sequences (SEQ ID NOs 3 and 5)
  • variant DNA molecules may be derived from the cDNA and gene sequences disclosed herein using standard DNA mutagenesis techniques as described above, or by synthesis of DNA sequences
  • this invention also encompasses nucleic acid sequences which encode a DIM-5 protein, but which vary from the disclosed nucleic acid sequences by virtue of the degeneracy of the genetic code
  • Variants of the DIM-5 protein may also be defined in terms of their sequence identity with the prototype DIM-5 protein shown in SEQ ID NOs 3 and 5
  • DIM-5 proteins share at least 50%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%
  • Nucleic acid molecules that are derived from the human DIM-5 encoding nucleic acid sequences disclosed include molecules that hybridize under stringent conditions to the disclosed prototypical DIM-5 nucleic acid molecules, or fragments thereof. Stringent conditions are hybridization at 65° C in 6 x SSC, 5 x Denhardt's solution, 0.5% SDS and 100 ⁇ g sheared salmon testes DNA, followed by 15-30 minute sequential washes at 65° C in 2 x SSC, 0.5% SDS, followed by 1 x SSC, 0.5% SDS and finally 0.2 x SSC, 0.5% SDS.
  • Low stringency hybridization conditions to detect less closely related homologs
  • the wash steps may be terminated after the first 2 x SSC wash.
  • DIM-5 encoding molecules including SEQ ID NOs: 1 , 2, and 4
  • orthologs and homologs of these sequences may be inco ⁇ orated into transformation or expression vectors.
  • DIM-5 neurospora DIM-5 encoding sequences
  • proteins or polypeptides encoded by the antisense strand of the DIM-5 cDNA can likewise be expressed.
  • the purified DIM-5 protein or polypeptide may be used for functional analyses, antibody production, diagnostics, and patient therapy.
  • DNA sequence of the DIM-5 cDNA and its antisense strand can be manipulated in studies to understand the expression of the gene and the function of its product.
  • Mutant forms of DIM-5 or homologous proteins from other species may be isolated based upon information contained herein, and may be studied in order to detect alteration in expression patterns in terms of relative quantities, tissue specificity and functional properties of the encoded mutant DIM-5 protein.
  • Partial or full-length cDNA sequences, which encode for the subject protein may be ligated into bacterial expression vectors.
  • Methods for expressing large amounts of protein from a cloned gene introduced into Escherichia coli (E. coli) may be utilized for the purification, localization and functional analysis of proteins. For example, fusion proteins consisting of amino terminal peptides encoded by a portion of the E.
  • coli lacZ or trpE gene linked to DIM-5 proteins may be used to prepare polyclonal and monoclonal antibodies against these proteins. Thereafter, these antibodies may be used to purify proteins by immunoaffinity chromatography, in diagnostic assays to quantitate the levels of protein and to localize proteins in tissues and individual cells by immunofluorescence. Intact native protein may also be produced in E. coli in large amounts for functional studies. Methods and plasmid vectors for producing fusion proteins and intact native proteins in bacteria are described in Sambrook et al. (Sambrook et al, In Molecular Cloning: A Laboratory Manual, Ch.
  • fusion proteins may be made in large amounts, are easy to purify, and can be used to elicit antibody response
  • Native proteins can be produced in bacteria by placing a strong, regulated promoter and an efficient ⁇ bosome-binding site upstream of the cloned gene If low levels of protein are produced, additional steps may be taken to increase protein production, if high levels of protein are produced, purification is relatively easy Suitable methods are presented in Sambrook et al (In Molecular Cloning A Laboratory Manual, CSHL, New York, 1989) and are well known in the art Often, proteins expressed at high levels are found in insoluble inclusion bodies Methods for extracting proteins from these aggregates are described by Sambrook et al (In Molecular Cloning A Laboratory Manual, Ch 17, CSHL, New York, 1989) Vector systems suitable for the expression of lacZ fusion genes include the pUR series of vectors (Ruther and Muller- Hill, EMBO J 2 1791, 1983), pEXl-3 (Stanley
  • the cDNA sequence may be ligated to heterologous promoters, such as the simian virus (SV) 40 promoter in the pSV2 vector (Mulligan and Berg, Proc Natl Acad Sci USA 78 2072-2076, 1981), and introduced into cells, such as monkey COS-1 cells (Gluzman, Cell 23 175-182, 1981), to achieve transient or long-term expression
  • heterologous promoters such as the simian virus (SV) 40 promoter in the pSV2 vector (Mulligan and Berg, Proc Natl Acad Sci USA 78 2072-2076, 1981)
  • SV simian virus
  • pSV2 vector simian virus 40 promoter in the pSV2 vector
  • cells such as monkey COS-1 cells (Gluzman, Cell 23 175-182, 1981)
  • the stable integration of the chimeric gene construct may be maintained in mammalian cells by biochemical selection, such as neomycin (Southern and Berg, J Mol Appl Genet
  • DNA sequences can be manipulated with standard procedures such as restriction enzyme digestion, fill-in with DNA polymerase, deletion by exonuclease, extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA sequences, site-directed sequence- alteration via single-stranded bacteriophage intermediate or with the use of specific oligonucleotides in combination with PCR
  • the cDNA sequence (or portions derived from it) or a mini gene (a cDNA with an intron and its own promoter) may be introduced into eukaryotic expression vectors by conventional techniques These vectors are designed to permit the transcription of the cDNA in eukaryotic cells by providing regulatory sequences that initiate and enhance the transcription of the cDNA and ensure its proper splicing and polyadenylation Vectors containing the promoter and enhancer regions of the SV40 or long terminal repeat (LTR) of the Rous Sarcoma virus and polyadenylation and splicing signal from SV40 are readily available (Mulligan et al , Proc Natl Acad Sci USA 78 1078-2076, 1981 , Gorman et al, Proc Natl Acad Sci USA 78 6777-6781 , 1982)
  • the level of expression ofthe cDN A can be manipulated with this type of vector, either by using promoters that have different activities (for example, the baculovirus pAC373
  • some vectors contain selectable markers such as the gpt (Mulligan and Berg, Proc Natl Acad Sci USA 78 2072-2076, 1981) or neo (Southern and Berg, J Mol Appl Genet 1 327-341, 1982) bacterial genes These selectable markers permit selection of transfected cells that exhibit stable, long-term expression ofthe vectors (and therefore the cDNA)
  • the vectors can be maintained in the cells as episomal, freely replicating entities by using regulatory elements of viruses such as papilloma (Sarver et al , Mol Cell Biol 1 486, 1981) or Epstein-Barr (Sugden et al , Mol Cell Biol 5 410, 1985)
  • one can also produce cell lines that have integrated the vector into genomic DNA Both of these types of cell lines produce the gene product on a continuous basis
  • the cDNA, or fragments thereof can be introduced by infection with virus vectors Systems are developed that use, for example, retroviruses (Bernstein et al ,
  • eukaryotic expression systems can be used for studies of DIM-5 encoding nucleic acids and mutant forms of these molecules, the DIM-5 protein and mutant forms of this protein Such uses include, for example, the identification of regulatory elements located in the 5' region ofthe DIM-5 gene on genomic clones that can be isolated from human genomic DNA libraries using the information contained in the present invention.
  • the eukaryotic expression systems may also be used to study the function ofthe normal complete protein, specific portions ofthe protein, or of naturally occurring or artificially produced mutant proteins.
  • the expression vectors containing the DIM-5 gene sequence or cDNA, or fragments or variants or mutants thereof can be introduced into human cells, mammalian cells from other species or non-mammalian cells as desired.
  • the choice of cell is determined by the pu ⁇ ose ofthe treatment.
  • monkey COS cells Gluzman, Cell 23:175-182, 1981
  • Chinese hamster ovary CHO
  • mouse NIH 3T3 fibroblasts or human fibroblasts or lymphoblasts may be used.
  • the present disclosure thus encompasses recombinant vectors that comprise all or part ofthe DIM-5 gene or cDNA sequences for expression in a suitable host.
  • the DIM-5 DNA is operatively linked in the vector to an expression control sequence in the recombinant DNA molecule so that the DIM-5 polypeptide can be expressed.
  • the expression control sequence may be selected from the group consisting of sequences that control the expression of genes of prokaryotic or eukaryotic cells and their viruses and combinations thereof.
  • the expression control sequence may be specifically selected from the group consisting ofthe lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the early and late promoters of SV40, promoters derived from polyoma, adenovirus, retrovirus, baculovirus and simian virus, the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, the promoter ofthe yeast alpha-mating factors and combinations thereof.
  • the host cell which may be transfected with the vector of this invention, may be selected from the group consisting of E. coli, Pseudomonas, Bacillus subtilis, Bacillus stearothermophilus or other bacilli; other bacteria; yeast; fungi; insect; mouse or other animal; or plant hosts; or human tissue cells.
  • DIM-5 protein fragments having therapeutic properties may be expressed in this manner also.
  • Monoclonal or polyclonal antibodies may be produced to either the normal DIM-5 protein or mutant forms of this protein (including for instance the specific mutant isolated and discussed herein), as well as to proteins or peptides encoded for by the reverse complement ofthe disclosed DIM-5 sequences.
  • antibodies raised against these proteins or peptides would specifically detect the protein or peptide with which the antibodies are generated. That is, an antibody generated to the DIM-5 protein or a fragment thereof would recognize and bind the DIM-5 protein and would not substantially recognize or bind to other proteins found in target cells.
  • an antibody specifically detects the DIM-5 protein is made by any one of a number of standard immunoassay methods; for instance, the Western blotting technique (Sambrook et al, In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989).
  • Western blotting technique Standard immunoassay methods
  • total cellular protein is extracted from human cells (for example, lymphocytes) and electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel.
  • the proteins are then transferred to a membrane (for example, nitrocellulose) by Western blotting, and the antibody preparation is incubated with the membrane.
  • an anti-mouse antibody conjugated to an enzyme such as alkaline phosphatase.
  • an enzyme such as alkaline phosphatase.
  • an alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production of a dense blue compound by immunolocalized alkaline phosphatase.
  • Antibodies that specifically detect the DIM-5 protein will, by this technique, be shown to bind to the DIM-5 protein band (which will be localized at a given position on the gel determined by its molecular weight). Non-specific binding ofthe antibody to other proteins may occur and may be detectable as a weak signal on the Western blot. The non-specific nature of this binding will be recognized by one skilled in the art by the weak signal obtained on the Western blot relative to the strong primary signal arising from the specific antibody-DIM-5 protein binding.
  • Substantially pure DIM-5 protein or protein fragment (peptide) suitable for use as an immunogen may be isolated from the transfected or transformed cells as described above. Concentration of protein or peptide in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms per milliliter. Monoclonal or polyclonal antibody to the protein can then be prepared as follows:
  • Monoclonal Antibody Production by Hybridoma Fusion Monoclonal antibody to epitopes ofthe DIM-5 protein identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256:495-497, 1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms ofthe selected protein over a period of a few weeks. The mouse is then sacrificed, and the antibody-producing cells ofthe spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess un-fused cells destroyed by growth ofthe system on selective media comprising aminopterin (HAT media).
  • HAT media aminopterin
  • the successfully fused cells are diluted and aliquots ofthe dilution placed in wells of a microtiter plate where growth ofthe culture is continued.
  • Antibody-producing clones are identified by detection of antibody in the supernatant fluid ofthe wells by immunoassay procedures, such as ELISA, as originally described by Engvall (Meth. Enzymol. 70:419-439, 1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988).
  • Polyclonal antiserum containing antibodies to heterogeneous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein, which can be unmodified or modified to enhance immunogenicity. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and may require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with either inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appear to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis et al. (J. Clin. Endocrinol Metab. 33:988-991 , 1971).
  • Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations ofthe antigen, begins to fall. See, for example, Ouchterlony et al. (In Handbook of Experimental Immunology, Wier, D. (ed.) chapter 19. Blackwell, 1973). Plateau concentration of antibody is usually in the range of about 0.1 to 0.2 mg/ml of serum (about 12 ⁇ M). Affinity ofthe antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher (Manual of Clinical Immunology, Ch. 42, 1980). Cs Antibodies Raised against Synthetic Peptides
  • a third approach to raising antibodies against DIM-5 encoded proteins or peptides is to use one or more synthetic peptides synthesized on a commercially available peptide synthesizer based upon the predicted amino acid sequence ofthe DIM-5 encoded protein or peptide.
  • polyclonal antibodies to specific peptides within DIM-5 are generated using well-known peptide-based injection techniques. Briefly, polyclonal antibodies are generated by injecting DIM-5 peptides into rabbits. D. Antibodies Raised by Injection of DIM-5-Encoding Sequence
  • Antibodies may be raised against proteins and peptides of DIM-5 by subcutaneous injection of a DNA vector that expresses the desired protein or peptide, or a fragment thereof, into laboratory animals, such as mice. Delivery ofthe recombinant vector into the animals may be achieved using a hand-held form ofthe Biolistic system (Sanford et al, Particulate Sci. Techno!. 5:27-37, 1987) as described by Tang et al. (Nature 356:152-154, 1992). Expression vectors suitable for this purpose may include those that express the DIM-5 encoding sequence under the transcriptional control of either the human ⁇ -actin promoter or the cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • Antibody preparations prepared according to any one of these protocols are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample; or for immunolocalization ofthe DIM-5 protein, for instance for studies of chromatin structure and regulation.
  • antibodies e.g., DIM-5-specif ⁇ c monoclonal antibodies
  • DIM-5-specif ⁇ c monoclonal antibodies can be humanized by methods known in the art.
  • Antibodies with a desired binding specificity can be commercially humanized (Scotgene, Scotland, UK; Oxford Molecular, Palo Alto, CA).
  • This disclosure includes methods of modifying DNA methylation of one or more target genes, or target regions within a genome, by influencing the activity of a histone methyltransferase.
  • inhibition of histone methylation is used to reduce the level of DNA methylation, thereby increasing the expression of one or more otherwise silenced genes.
  • enhancement of histone methylation is used to increase the level of DNA methylation, thereby decreasing the expression of one or more target genes.
  • the activity of a HMTase in a cell can be influenced in any one of myriad ways, including increasing or decreasing the expression of a native HMTase; providing an additional copy of a native or heterologous HMTase (under control of, for instance, a constitutive or regulatable promoter) to a cell to increase the amount of HMTase expressed therein; providing a HMTase antisense or other suppressive construct (e.g., siRNA, dominant negative constructs, and so forth) to the cell to reduce the amount of HMTase expressed therein; applying recombinant purified HMTase to the cell; applying one or more agents that inhibit the activity of a HMTase to the cell (for instance, by competitive binding), thereby reducing the activity ofthe HMTase in the cell; applying one or more agents that increase the activity of a HMTase (for instance, by increasing the affinity ofthe HMTase for its target histone residue(s)); and so forth.
  • the histone methylation that is influenced is a methylation ofthe lysine 9 position of histone H3. It is believed that, as with other amino acid residues in target proteins, up to three methyl groups can modify this one amino acid residue. Therefore, it is contemplated that the methylation which is influenced or modified (e.g., inhibited) as described herein may be a first methylation, a second methylation, or a third methylation, or any combination thereof.
  • DIM-5 histone methyltransferase, which is essential for DNA methylation, trimethylates H3 lysine 9.
  • DIM-5 can generate mono- di-, and, especially, trimethylated species.
  • Chromatin immunoprecipitation experiments revealed trimethyl-lysine 9, but not dimethyl-lysine 9, associated with methylated DNA in Neurospora and dimethyl-lysine 4 preferentially associated with active genes. Elimination of DNA methylation by mutation ofthe DNA methyltransferase gene, dim-2, did not prevent trimethylation of lysine 9 but mutation of dim-5 did, suggesting that trimethylation of histone H3 lysine 9 directs DNA methylation in Neurospora.
  • Lysine residues such as lysine 9 in histone H3 may be modified in various ways, e.g., by the addition of acetyl groups (by histone acetytransferases, also known as HDACs) or by the addition of one, two, or three methyl groups.
  • This disclosure describes for the first time, a histone methytransferase (DIM-5) that efficiently trimethylates a lysine residue in a histone, which is illustrated by the demonstration that a dimethyl-K9 peptide (based on the sequence of histone H3 tails) is an excellent substrate for DIM-5.
  • DIM-5 histone methytransferase
  • DNA methylation-related diseases may be associated with alterations in the degree of methylation (0, 1, 2, or 3 methyl groups) on K 9 of histone H3, or on one or more other residues in histone H3 or another histone molecule.
  • this disclosure provides methods to assay the degree of methylation. Also provided are methods to treat DNA methylation-related diseases by specifically affecting histone MTases specific for a particular methylation degree (e.g., trimethyl). With respect to characterization ofthe extent of methylation of a particular residue such as
  • methylation- degree specific antibodies can be used as reagents to recognize chromatin defects that lead to defects in DNA methylation.
  • This disclosure therefore further includes methods of determining whether a subject is suffering from, or is likely to develop, a DNA methylation-related disease or condition, which methods involve determining the amount, extent, number (e.g., first, second, or third methylation on a single residue) or position (e.g., on which residue) of histone methylation in a cell ofthe subject.
  • One of ordinary skill in the art will know methods for assessing these qualities regarding the methylation of specific target proteins. Specific examples of certain methylation detection methods are provided herein, for instance in Example 1.
  • tri-methylation specifically is assessed, using for instance an antibody specific for the tri-methyl form of a specific target methylated residue in a histone.
  • an antibody specific for the tri-methyl modified residue at position lysine 9 of histone H3 can be used to determine the extent of tri-methylation of this residue in a cell.
  • such antibodies can be used to perform a chromatin immunoprecipitation, and the precipitated material analyzed for instance to determine what regions ofthe genome are associated with methylated histones.
  • a HMTase (such as DIM-5) is used in a method to assess the potential for a cell to accept histone methylation that is correlated with the potential for developing a methylation-related condition or disease.
  • the HMTase is contacted with a cell, or a nuclear or chromatin preparation ofthe cell, in the presence of a detectable methyl group donor (for instance, labeled with an isotope or fluorescent tag) under conditions in which the HMTase can methylate appropriate available targets.
  • a detectable methyl group donor for instance, labeled with an isotope or fluorescent tag
  • the sample is analyzed to determine where and/or to what extent the HMTase has methylated one or more molecules in the sample.
  • the extent of labeled methylation is determined for the entire sample (after the sample is washed to remove uninco ⁇ orated label).
  • the sample is analyzed to see if label is inco ⁇ orated at one or more specific sites, for instance one or more specific residues on a histone molecule.
  • the reaction is run in the presence of at least one DMTase, and the resultant sample is further analyzed for the amount and or location of inco ⁇ oration of labeled methyl groups into one or more DNA target sequences.
  • cancer cells or potentially cancerous or precancerous cells
  • the data gathered from such analyses is then used to predict the likelihood of cancer development or progression, efficacy of treatment, to aid in the selection of treatment, and/or to diagnose whether cancer is present.
  • This disclosure further relates in some embodiments to novel methods for screening test compounds for their ability to treat, detect, analyze, ameliorate, reverse, and/or prevent a methylation-related disease or condition, especially neoplasia and pre-cancerous lesions.
  • the present disclosure provides methods for identifying test compounds that can be used to treat, ameliorate, reverse, and/or prevent neoplasia, including pre-cancerous lesions.
  • the compounds of interest can be tested by exposing the novel HMTase described herein to the compounds, and if a compound inhibits this novel HMTase, the compound is then further evaluated for its anti-neoplastic properties.
  • One aspect involves a screening method to identify a compound effective for treating, preventing, or ameliorating a methylation-related disease or condition, especially neoplasia, which method includes ascertaining the compound's inhibition of this novel HMTase or another HMTase.
  • the screening method further includes determining whether the compound inhibits the growth of tumor cells in a cell culture.
  • Histone methyltransferases for instance the novel HMTase DIM-5 and homologs and orthologs of this molecule, are useful to identify compounds that can be used to treat, ameliorate, or prevent a methylation-related disease or condition, such as neoplasms.
  • the screening or creation, identification and selection of appropriate high affinity inhibitors of histone methyltransferases can be accomplished by a variety of methods. Broadly speaking these may include, but are not limited to two general approaches. One approach is to use structural knowledge about the target enzyme to design a candidate molecule with which it will precisely interact. Examples include computer assisted molecular design and protein crystallographic studies. Specific examples of certain protein crystallographic studies are provided herein, for instance in Example 11. A second approach is to use combinatorial or other libraries of molecules, whereby a large library of molecules is screened for affinity with regard to the target enzyme.
  • Cancer and precancer may be thought of as diseases that involve unregulated cell growth.
  • Cell growth involves a number of different factors. One factor is how rapidly cells proliferate, and another involves how rapidly cells die. Cells can die either by necrosis or apoptosis depending on the type of environmental stimuli. Cell differentiation is yet another factor that influences tumor growth kinetics. Resolving which ofthe many aspects of cell growth a test compound affects can be important to the discovery of a relevant target for pharmaceutical therapy. Screening assays based on this technology can be combined with other tests to determine which compounds have growth inhibiting and pro-apoptotic activity.
  • Some embodiments provided herein involve determining the histone methyltransferase inhibition activity of a given compound, for instance an H3 histone methyltransferase inhibition activity.
  • Test compounds can be assessed for their probable ability to treat neoplastic lesions either directly, or indirectly by comparing their activities against compounds known to be useful for treating neoplasia.
  • Methods are provided herein for determining the methylation level of a target protein, such as histone H3. These methods can be used to determine the effectiveness of test compounds for inhibiting methylation. Other methods for determining methylation of proteins or specific residues within proteins will be known to those of ordinary skill in the art.
  • Compounds can be screened for inhibitory or other effects on the activity ofthe novel histone methyltransferase DIM-5 described herein (or on another H3 histone methyltransferase such as clr4 or su(var)3-9 or another homolog) using an expressed recombinant version ofthe enzyme, or a homolog or ortholog isolated from another species, for instance a mammal such as a human.
  • cells expressing one of these HMTases can be treated with a test compound and the effect ofthe test compound on methylation of a specific methylation target (e.g., K9 of histone H3) can be determined, for instance using one ofthe techniques described herein. Additional detail regarding methods for determining histone methylation influencing activity (e.g., inhibition) is provided herein.
  • provided screening methods involve further determining whether the compound reduces the growth of tumor cells.
  • Various cell lines can be used, which may be selected based on the tissue to be tested.
  • these cell lines include: SW-480 - colonic adenocarcinoma; HT-29 - colonic adenocarcinoma, A-427 - lung adenocarcinoma carcinoma; MCF- 7 - breast adenocarcinoma; and UACC-375 - melanoma line; and DU145 - prostrate carcinoma. Cytotoxicity data obtained using these cell lines are indicative of an inhibitory effect on neoplastic lesions. These cell lines are well characterized, and are used for instance by the United States National Cancer Institute (NCI) in their screening program for new anti-cancer drugs.
  • NCI National Cancer Institute
  • a test compound's ability to inhibit tumor cell growth in vitro can be measured using the HT-29 human colon carcinoma cell line obtained from ATCC (Bethesda, MD).
  • HT-29 cells have previously been characterized as a relevant colon tumor cell culture model (Fogh & Trempe, In: Human Tumor Cells in Vitro, Fogh (ed.), Plenum Press, N.Y., pp. 115-159, 1975).
  • HT- 29 cells are maintained in RPMI media supplemented with 5% fetal bovine calf serum (Gemini Bioproducts, Inc., Carlsbad, Calif.) and 2 mM glutamine, and 1% antibiotic-antimycotic, in a humidified atmosphere of 95% air and 5% C0 2 at 37° C. Briefly, HT-29 cells are plated at a density of 500 cells/well in 96 well microtiter plates and incubated for 24 hours at 37° C. prior to the addition of test compound. Each determination of cell number involved six replicates.
  • the cells After six days in culture, the cells are fixed by the addition of cold trichloroacetic acid (TCA) to a final concentration of 10% and protein levels are measured, for instance using the sulforhodamine B (SRB) colorimetric protein stain assay as previously described by Skehan et al. (J. Natl. Cancer Inst. 82: 1 107- 1 12, 1990).
  • TCA cold trichloroacetic acid
  • SRB sulforhodamine B
  • a number of other methods are available to measure growth inhibition and could be substituted for the SRB assay. These methods include counting viable cells following trypan blue staining, labeling cells capable of DNA synthesis with BrdU or radiolabeled thymidine, neutral red staining of viable cells, or MTT staining of viable cells.
  • IC 50 value may be determined and used for comparative pu ⁇ oses. This value is the concentration of drug needed to inhibit tumor cell growth by 50% relative to the control. In some embodiments, the IC 50 value is less than 100 ⁇ M in order for the compound to be considered further for potential use for treating, ameliorating, or preventing neoplastic lesions.
  • screening methods provided herein further involve determining whether the test compound induces apoptosis in cultures of tumor cells.
  • necrosis and apoptosis Two distinct forms of cell death may be described by mo ⁇ hological and biochemical criteria: necrosis and apoptosis. Necrosis is accompanied by increased permeability ofthe plasma membrane, whereby the cells swell and the plasma membrane ruptures within minutes. Apoptosis is characterized by membrane blebbing, condensation of cytoplasm, and the activation of endogenous endonucleases.
  • Apoptosis occurs naturally during normal tissue turnover and during embryonic development of organs and limbs. Apoptosis also can be induced by various stimuli, including cytotoxic T-lymphocytes and natural killer cells, by ionizing radiation and by certain chemotherapeutic drugs. Inappropriate regulation of apoptosis is thought to play an important role in many pathological conditions including cancer, AIDS, or Alzheimer's disease, etc.
  • Test compounds can be screened for induction of apoptosis using cultures of tumor cells maintained under conditions as described above.
  • treatment of cells with test compounds involves either pre- or post-confluent cultures and treatment for two to seven days at various concentrations ofthe test compounds.
  • Apoptotic cells can be measured in both the attached and "floating" portions ofthe cultures. Both are collected by removing the supernatant, trypsinizing the attached cells, and combining both preparations following a centrifugation wash step (10 minutes, 2000 ⁇ m).
  • cultures can be assayed for apoptosis and necrosis, for instance by florescent microscopy following labeling with acridine orange and ethidium bromide.
  • Many methods for measuring apoptotic cells are known to those of ordinary skill in the art; for instance, one method for measuring apoptotic cell number has been described by Duke & Cohen (Curr. Prot. Immuno., Coligan et al, eds., 3.17.1-3.17.1, 1992). For example, floating and attached cells are collected by trypsinization and washed three times in PBS. Aliquots of cells are then centrifuged.
  • the pellet is resuspended in media and a dye mixture containing acridine orange and ethidium bromide prepared in PBS and mixed gently. The mixture then can be placed on a microscope slide and examined for mo ⁇ hological features of apoptosis.
  • Apoptosis also can be quantified by measuring an increase in DNA fragmentation in cells that have been treated with test compounds.
  • Commercial photometric EIA for the quantitative in vitro determination of cytoplasmic histone-associated-DNA-fragments are available (e.g., Cell Death Detection ELISA, Boehringer Mannheim).
  • the Boehringer Mannheim assay is based on a sandwich-enzyme-immunoassay principle using mouse monoclonal antibodies directed against DNA and histones, respectively. This allows the specific determination of mono- and oligo-nucleosomes in the cytoplasmic fraction of cell lysates.
  • apoptosis is measured as follows: The sample (cell-lysate) is placed into a streptavidin-coated microtiter plate ("MTP"). Subsequently, a mixture of anti-histone-biotin and anti-DNA peroxidase conjugates is added and incubated for two hours. During the incubation period, the anti-histone antibody binds to the histone-component ofthe nucleosomes and simultaneously fixes the immunocomplex to the streptavidin-coated MTP via its biotinylation. Additionally, the anti- DNA peroxidase antibody reacts with the DNA component ofthe nucleosomes.
  • MTP streptavidin-coated microtiter plate
  • Peroxidase is determined photometrically with ABTS7 (2,2'-Azido- [3-ethylbenzthiazolin-sulfonate]) as substrate.
  • SW-480 colon adenocarcinoma cells are plated in a 96-well MTP at a density of 10,000 cells per well. Cells are then treated with test compound, and allowed to incubate for 48 hours at 37° C. After the incubation, the MTP is centrifuged and the supernatant is removed. The cell pellet in each well is then resuspended in lysis buffer for 30 minutes. The lysates are then centrifuged and aliquots ofthe supernatant (i.e., cytoplasmic fraction) are transferred into a streptavidin-coated MTP.
  • the supernatant i.e., cytoplasmic fraction
  • EC 50 values may also be determined by evaluating a series of concentrations of the test compound.
  • apoptosis i.e., greater than two fold stimulation at a test compound concentration of 100 ⁇ M
  • the EC 50 value for apoptotic activity should be less than 100 ⁇ M for the compound to be further considered for potential use for treating neoplastic lesions.
  • EC 50 is understood herein to be the concentration that causes 50% induction of apoptosis relative to vehicle treatment.
  • Test compounds identified by the methods described herein can be tested for antineoplastic activity by their ability to inhibit the incidence of preneoplastic lesions in an organ culture system, such as a mammary gland organ culture system.
  • organ culture system such as a mammary gland organ culture system.
  • the mouse mammary gland organ culture technique has been successfully used by other investigators to study the effects of known antineoplastic agents such as NSAIDs, retinoids, tamoxifen, selenium, and certain natural products, and is useful for validation ofthe screening methods provided herein.
  • female BALB/c mice can be treated with a combination of estradiol and progesterone daily, in order to prime the glands to be responsive to hormones in vitro.
  • the animals are sacrificed, and thoracic mammary glands are excised aseptically and incubated for ten days in growth media supplemented with insulin, prolactin, hydrocortisone, and aldosterone.
  • DMBA 7,12- dimethylbenz(a)anthracene
  • Fully developed glands are then deprived of prolactin, hydrocortisone, and aldosterone, resulting in the regression ofthe glands but not the premalignant lesions.
  • test compound is dissolved in, for instance, DMSO and added to the culture media for the duration ofthe culture period.
  • the glands are fixed in 10% formalin, stained with alum carmine, and mounted on glass slides.
  • the incidence of forming mammary lesions is the ratio ofthe glands with mammary lesions to glands without lesions.
  • the incidence of mammary lesions in test compound treated glands is compared with that ofthe untreated glands.
  • the extent ofthe area occupied by the mammary lesions can be quantitated by projecting an image ofthe gland onto a digitation pad.
  • the area covered by the gland is traced on the pad and considered as 100% ofthe area.
  • the space covered by each ofthe unregressed structures is also outlined on the digitization pad and quantitated by the computer.
  • Hypermethylation-associated diseases, disorders, and conditions are characterized by exhibiting hypermethylation of one or more DNA sequences. Such diseases, disorders, and conditions therefore can be identified by examining the methylation state (or level) of nucleic acids in a subject known to or suspected of suffering therefrom; a high level of specific or general DNA methylation indicates that the disease/disorder/condition is hypermethylation-associated. It is beneficial to treat (or prevent) such diseases, disorders, and conditions with compounds that influence (e.g., inhibit) an activity of a HMTase.
  • the compound is provided in the form of a pharmaceutical composition.
  • subjects prior to administration of a HMTase-inhibiting compound, subjects will be screened to find those whose condition involves hypermethylation of one or more DNA sequences, and thus are most likely to be susceptible to treatment with an HMTase inhibitor.
  • screening in some embodiments involves examining the methylation level ofthe genome of cell or tissue sample from the subject, or of a specific target sequence from such genome, or of a specific target protein such as H3 histone, or a specific amino acid residue of a target protein, or some combination of two or more of these.
  • HMTase methylation presumably can be used to influence these processes by altering the DNA methylation state ofthe system.
  • hypomethylation activity of HMTase inhibitors can be used to reduce antimicrobial resistance, similarly to the system described in United States Patent No. 5,872,104 (entitled “Combinations and Methods for Reducing Antimicrobial Resistance”). Examples of such methods work by reducing the methylation-mediated binding inhibition of an antibiotic agent, for instance on an rRNA molecule, thereby increasing the susceptibility ofthe treated microbes to that antibiotic agent.
  • the HMTase described herein influences the methylation of DNA, it can be useful to be able to detect and/or quantify DNA methylation for use with one or more aspects ofthe methods and compositions disclosed herein. Though specific examples of detection and quantification methods are provided, those of ordinary skill in the art are familiar with other methods that could be used.
  • Each RE can "cut" DNA at a certain short (e.g., 4-8 nucleotide) recognition sequence. The position of such cuts can be determined based on the length of fragments produced after a digestion reaction, which fragments are detected, for instance, by gel electrophoresis, transfer to a membrane and hybridization.
  • Certain REs are "methylation-sensitive" in that certain bases within the recognition sequence must be unmethylated for digestion to occur. Examples of methylation-sensitive REs include S ⁇ w3AI and Dpn .
  • the band pattern after digestion with a methylation-sensitive RE changes depending on the methylation pattern ofthe DNA.
  • Techniques based on methylation-sensitive REs can be somewhat limited, because many CpG's that might be methylated are outside the recognition sequences of REs, and thus cannot be examined using these methods. Methods also are available to examine individual potential methylation sites. See, for instance, Shemer e/ ⁇ /. (PNAS 93:6371-6376, 1996) and Kafri et al. (Genes Dev. 6:705-714, 1992), which describe a PCR-based method to detect methylation in a specific target sequence.
  • Ms-SNuPE Methylation-sensitive Single Nucleotide Primer Extension
  • unmethylated cytosines (C or T) is determined by incubating the annealed product with Taq polymerase and either (a- 32 P) dCTP or (a- 32 P) dTTP, followed by gel electrophoresis and Phosphorlmager analysis.
  • High-throughput methylation assays are also useful for measuring methylation.
  • one such assay is the Methylight assay (Eads et al, Cancer Res. 61:3410-3418, 2001 ; published international patent application PCT US00/13029), a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (TaqMan) technology.
  • the patent literature is also replete with methods for detecting and/or measuring methylation in a nucleic acid molecule. See, for instance:
  • DNA methylation quantitation and detection methods are illustrated in the Examples, below.
  • the present disclosure also includes methods of treatment for methylation-mediated disease, such as a hyper-proliferative disease or disorder, in a subject.
  • the method includes administering an HMTase-inhibitory compound, or an analog, mimetic, prodrug, or derivative thereof that has similar hypomethylation function, or a combination of such compound and one or more other pharmaceutical agents, to the subject in a pharmaceutically compatible carrier and in an amount effective to inhibit the development or progression of a methylation-mediated disease.
  • subjects can also be selected using more specific criteria, such as a definitive diagnosis ofthe disease/condition or identification of one or more factors that increase the likelihood of developing such disease (e.g., a genetic, environmental, or lifestyle factor).
  • histone methylation is an example of a stable modification of histones and is demonstrated herein as influencing DNA methylation
  • limited treatments to cause DNA hypomethylation e.g., treatment with 5-azacytidine or zebularine
  • DNA hypomethylation could fail if the underlying methylation of histone H3 is not changed. It is believed that in some circumstances it is more effective to target histone methylation in treatments that aim to change DNA methylation in a stable, or relatively stable, way, rather than attempting to alter (e.g., inhibit or block) DNA methylation directly.
  • the vehicle in which the drug is delivered can include pharmaceutically acceptable compositions ofthe compounds, using methods well known to those with skill in the art. Any ofthe common carriers, such as sterile saline or glucose solution, can be utilized. Routes of administration include but are not limited to oral and parenteral routes, such as intrathecal, intravenous (iv), intraperitoneal (ip), rectal, topical, ophthalmic, nasal, and transdermal.
  • routes of administration include but are not limited to oral and parenteral routes, such as intrathecal, intravenous (iv), intraperitoneal (ip), rectal, topical, ophthalmic, nasal, and transdermal.
  • the compounds may be administered intravenously in any conventional medium for intravenous injection, such as an aqueous saline medium, or in blood plasma medium.
  • the medium may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, lipid carriers such as cyclodextrins, proteins such as serum albumin, hydrophilic agents such as methyl cellulose, detergents, buffers, preservatives and the like.
  • pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, lipid carriers such as cyclodextrins, proteins such as serum albumin, hydrophilic agents such as methyl cellulose, detergents, buffers, preservatives and the like.
  • compositions in some embodiments can be prepared with conventional pharmaceutically acceptable carriers, adjuvants, and counter-ions as would be known to those of skill in the art.
  • the compositions in some embodiments are in the form of a unit dose in solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions, or suspensions.
  • the compounds ofthe present disclosure can be administered at about the same dose throughout a treatment period, in an escalating dose regimen, or in a loading-dose regime (e.g., in which the loading dose is about two to five times the maintenance dose).
  • the dose is varied during the course of a treatment based on the condition ofthe subject being treated, the severity ofthe disease or condition, the apparent response to the therapy, and/or other factors as judged by one of ordinary skill in the art.
  • long-term treatment with the drug is contemplated, for instance in order to reduce the occurrence of remethylation of a tumor suppressor gene.
  • sustained intra-tumoral (or near-tumoral) release ofthe pharmaceutical preparation that comprises a hypomethylation effective amount of a HMTase inhibitor may be beneficial.
  • a hypomethylation effective amount of a HMTase inhibitor may be beneficial.
  • polymers such as bis(p-carboxyphenoxy)propane-sebacic-acid or lecithin suspensions may be used to provide sustained intra-tumoral release.
  • delivery is via an injected and/or implanted drug depot, for instance comprising multi-vesicular liposomes such as in DepoFoam (SkyePharma, Inc, San Diego, CA) (see, for instance, Chamberlain et al, Arch. Neuro. 50:261-264, 1993; Katri et al, J. Pharm. Sci. 87: 1341-1346, 1998; Ye et al, J. Control Release 64: 155-166, 2000; and Howell, Cancer . 7:219-227, 2001 ).
  • DepoFoam Stema, Inc, San Diego, CA
  • perfusion of a tumor with a pharmaceutical composition that contains a hypomethylation effective amount of a HMTase-inhibitory compound is contemplated.
  • Therapeutically effective doses ofthe compounds ofthe present disclosure can be determined by one of skill in the art. Low toxicity of certain identified compounds makes it possible to administer high doses, for example 100 mg/kg, although doses of 10 mg/kg, 20 mg/kg, 30 mg/kg or more are contemplated, though lower dosages are also contemplated.
  • An example of a dosage range is 0.1 to 200 mg/kg body weight orally in single or divided doses.
  • Another example of a dosage range is 1.0 to 100 mg/kg body weight orally in single or divided doses.
  • compositions are, for example, provided in the form of a tablet containing 0.01 to 1000 mg ofthe active ingredient, particularly 1, 5, 10, 15, 20, 25, 50, 100, 200, 400, 500, 600, 800, and 1000 mg ofthe active ingredient for the symptomatic adjustment ofthe dosage to the subject being treated.
  • the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity ofthe specific compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, and severity ofthe condition ofthe host undergoing therapy.
  • the pharmaceutical compositions comprising a hypomethylation effective amount of at least one HMTase inhibitor can be used in the treatment or prevention of a variety of diseases and conditions that are associated with and/or caused by hypermethylation of one or more specific gene sequences.
  • cancers in particular tumors that are characterized by having one or more hypermethylated sequences such as a tumor suppressor gene, particularly where the hypermethylation has resulted in the inactivation (silencing) of that gene.
  • inactivated genes and associated cancers have now been identified, including for instance: cadherin (inactivation of which is often associated with breast or prostate tumors and squamous cell lung carcinoma); estrogen receptor (inactivation of which is often associated with estrogen receptor negative breast tumors); VHL (inactivation of which is associated with renal cancer); H19 (a tumor suppressor gene located on 1 lp, the inactivation of which is implicated in many tumors); 14-3-3 ⁇ (silenced in some breast cancers); Apaf-1 (inactivated in metastatic melanomas, though it appears that the methylation inactivation related to this gene may be indirect or through a genetic region other than the Apaf-l promoter); and p53 (a tumor suppressor gene, the inactivation of which is
  • GST glutathione-S-transferase
  • TIMP-3 tissue inhibitor of metalloproteinase-3
  • methylcytidine in the genome can lead to mutation.
  • some cancers arise from or are enhanced by mutations in genes where the mutation is thought to have been caused by methylation of a cytidine residue, followed by the subsequent conversion ofthe methylated cytidine to a guanidine. This can result in tumor gene destabilization, tumor metastasis, tumor progression, tumor recurrence, and resistance ofthe tumor to therapy by cytotoxic agents.
  • Sub- clones ofthe tumor containing the mutated gene(s) may be more aggressive, metastatic, and therapy resistant. It is believed that a HMTase inhibitor DNA hypomethylation activity may be used to prevent or reduce the likelihood of such mutations.
  • the present disclosure also contemplates combinations of one or more HMTase inhibitory compounds with one or more other agents useful in the treatment of hypermethylation-related disease.
  • the compounds of this disclosure may be administered in combination with effective doses of other medicinal and pharmaceutical agents.
  • one or more known anti-cancer drugs are included with the HMTase inhibitor.
  • administration in combination with refers to both concurrent and sequential administration ofthe active agents.
  • the compounds and/or peptides of this invention may be administered in combination with effective doses of radiation, anti-proliferative agents, anti-cancer agents, direct DNA methylation inhibitors, immunomodulators, anti-inflammatories, anti-infectives, hypomethylation agents, nucleosides and analogs thereof, and/or vaccines.
  • anti-proliferative agents that can be used in combination with a HMTase inhibitor as provided herein include, but are not limited to, the following: ifosamide, cisplatin, methotrexate, procarizine, etoposide, BCNU, vincristine, vinblastine, cyclophosphamide, gencitabine, 5-fluorouracil, paclitaxel, or doxorubicin.
  • Non-limiting examples of immuno-modulators that can be used in combination with a HMTase inhibitor as provided herein are AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New La, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).
  • HMTase inhibitor examples include 5-azacytidine, Zebularine, 2'-deoxy-4-azacytidine, ara-C, and tricostatin A. It is believed that such agents may be additive and or synergistic with the HMTase inhibitor in inhibiting DNA methylation.
  • the combination therapies are of course not limited to the lists provided in these examples, but includes any composition for the treatment of diseases or conditions associated with hypermethylation of one or more gene sequences.
  • kits for use in inhibiting a DNA methylation kits for use in reducing the methylation of a histone or a nucleic acid, and kits for prevention and/or treatment of a disorder, condition or diseases (e.g., a hyper-proliferative disorder, such as neoplasm, in particular a hyper-proliferative disorder that is mediated by methylation of one or more gene sequences).
  • a hypomethylating effective amount of one or more ofthe compounds is provided in one or more containers.
  • the compounds may be provided suspended in an aqueous solution or as a freeze-dried or lyophilized powder, for instance.
  • kits can also include instructions, usually written instructions, to assist the user in treating or preventing a disorder, condition or disease (e.g., a methylation-mediated hyper-proliferative disorder) with a HMTase-activity modifying compound and/or binding peptide.
  • a disorder, condition or disease e.g., a methylation-mediated hyper-proliferative disorder
  • Such instructions can optionally be provided on a computer readable medium.
  • the container(s) in which the compound(s) are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, ampoules, or bottles.
  • the therapeutic compound may be provided in pre-measured single use amounts in individual, typically disposable, tubes, or other such containers.
  • the amount of a compound supplied in the kit can be any appropriate amount, depending for instance on the market to which the product is directed. For instance, if the kit were adapted for research or clinical use, the amount of each HMTase-activity modifying compound provided likely would be an amount sufficient for several treatments.
  • kits will also include one or more other agents useful treating or preventing a disease or condition, for instance an agent useful in directly inhibiting DNA methylation, or another agent useful in inhibiting cell proliferation that is mediated by or influenced by hypermethylation of a gene sequence, e.g. in treating hyper-proliferation of a methylation-associated tumor.
  • agents useful treating or preventing a disease or condition for instance an agent useful in directly inhibiting DNA methylation, or another agent useful in inhibiting cell proliferation that is mediated by or influenced by hypermethylation of a gene sequence, e.g. in treating hyper-proliferation of a methylation-associated tumor.
  • such kits may include one or more effective doses of anti-proliferative or anti-cancer drugs.
  • Genomic DNA was isolated from liquid cultures grown two days at 32° C and analyzed for DNA methylation by Southern hybridization as previously described (Foss et al, Science 262:1737- 1741, 1993).
  • the probe for the ⁇ 63 region was a 0.9 kb Ban ⁇ l-EcoO ⁇ 09 fragment isolated from pPG22 (Margolin et al, Genetics 149: 1787-1797, 1998).
  • the 0.8 kb BamHl fragment was used to probe for the ⁇ - ⁇ region and a 9.2 kb Kpnl fragment representing one repeat unit ofthe rDNA was used was used to probe for rDNA.
  • the 1 D21 and 9a20 probes were generated by PCR from the wild- type strain 74-OR23-IVA.
  • Strains used for the methylation analysis shown in FIG 2 were: 74-OR23- IVA (wt; all blots), dim-2 strains N1275 (Foss et al, Science 262:1737-1741, 1993) (dim-2 arg-10 mat A; ⁇ 63, ⁇ - ⁇ and rDNA blots) and N1877 (Kouzminova & Selker, EMBO Journal 20:4309-4323, 2001) (dim-2::hph his-3 mat a; 1D21 and 9A20 blots) and dim-5 strains N2144 (dim-5; ⁇ 63, ⁇ - ⁇ and rDNA blots); N2145 (dim-5 leu-2 pan-2 mat a; 1 D21 blot) and N2140 (dim-5 leu-2 pan-2 mat A; 9A20 blot).
  • PCR fragment containing the presumed dim-5 ORF was generated, cut within the SET domain ofthe gene with EcoRV, and introduced into strain N644 (Irelan & Selker, Genetics 146:509-523, 1997; dim-5 + am WP lhph/am p am inl mat a) by co-transformation with pBT6.
  • the PCR product was digested with BamHl and EcoP ⁇ , gel-purified and cloned into the GST- fusion expression vector pGEX-5X-3 (Pharmacia) using E. coli strain DH5ocF'.
  • Recombinant protein was prepared from a 800 ml culture of E.
  • coli cells grown three hours at 37° C in LB medium with ampicillin (400 ⁇ g/ml), shifted to 30° C for 40 minutes, induced with IPTG (0.1 mM) and collected one hour later.
  • Cells were lysed by sonication on ice in 8 ml of RIPA buffer [20 mM Tris (pH 7.5), 500 mM NaCl, 5 mM EDTA, 1% IGEPAL CA-630 (Sigma), 0.5% deoxycholate] (Rea et al, Nature 406:593-599, 2000) with 5 mg/ml lysozyme and a proteinase inhibitor cocktail (CompleteTM;
  • HMTase assays were carried out on a natural mixture of calf thymus histones as described (Rea et al, Nature 406:593-599, 2000) except that the reaction was carried out at 20° C for 6 hours with 2.75 ⁇ Ci S-adenosyl-[methyl- 3 H]-L-methionine (0.55 mCi/ml; NEN). Products were fractionated on SDS-polyacrylamide (16.5%; 29:1) gels and fluorographed (4-12 hours) using ENTENSIFYTM (DuPont).
  • Example 2 Isolation and Genetic Mapping of the dim-5 Mutation
  • the interval between the putative leu-2 and trp-4 genes was scrutinized for dim-5 candidates.
  • One of 15 candidates in this region identified using BLASTx is predicted to encode a protein related to chromatin-associated proteins involved in gene silencing in fission yeast and fruit flies, namely Schizosaccharomyces pombe Clr4 (Ivanova et al, Nat Genet 19: 192-195, 1998) and Drosophila melanogaster Su(var)3-9 (Jschiersch et al, EMBO J. 13:3822-3831, 1994).
  • Example 4 De-repression of a Silenced Transgene by Banling dim-5 It remained formally possible that ectopic insertions ofthe Neurospora clr4/ su(var)3-9 homologue suppressed the dim-5 mutation as the result of a dosage effect but was not itself C/ /M-5. We took advantage of "quelling," a post-transcriptional gene silencing mechanism of Neurospora that does not depend on DNA methylation (Cogoni et al, Embo J 15:3153-3163, 1996) to carry out an independent test ofthe clr4/su(var)3-9 homologue.
  • Example 5 The dim-5 Mutant has a Nonsense Mutation in the Evolutiona ⁇ ly
  • DIM-5 is a SET domain protein homologous to genes required for heterochromatin formation
  • the SET domain was initially identified as a region of apparent homology in three nuclear proteins of Drosophila, Su(var)3-9, the polycomb group protein E(Z) and t ⁇ thorax-group protein TRX (Tschiersch et al , EMBO J 3 3822-3831 , 1994) Greater than 200 genes with SET domains are now known (Jenuwein et al , Trends Cell Biol 1 1 266-273, 2001) Like clr4, su(var)3-9
  • DIM-5 is a histone methyltransferase
  • GST glutathione-S-transferase
  • Recombinant DIM-5 fusion protein was purified from E coli, provided with S-adenosyl-[methyl- 3 H]-L-meth ⁇ on ⁇ ne as a potential methyl-group donor and incubated with a natural mixture of histones from calf thymus
  • the proteins were then fractionated by SDS-polyacrylamide gel electrophoresis and assayed for inco ⁇ oration of methyl groups by fluorography and scintillation counting of gel slices Significant incorporation of labeled methyl groups into histones was detected, indicating that DIM-5 is a bonafide H
  • hH3 gene was mutated in vitro, replacing the lysine codon with codons for leucine (L) or arginine (R), and the modified genes were introduced into strain N644 using co-transformation
  • Leucine and arginine were chosen because (1) they are structurally similar to lysine, (2) the neutral amino acid leucine can be regarded as a mimic of an acetylated lysine, (3) the positively charged amino acid arginine can be regarded as a mimic of an unacetylated lysine, and (4) leucine is known not to be a substrate for methylation of recombinant Suv39hl HMTase (Rea et al , Nature 406 593-599, 2000)
  • Lysine 9 ofN crassa histone H3 (Woudt et al , Nucl Acids Res 1 1 5347-5361 , 1983) was changed to leucine and arginine using the PCR-based QuickChangeTM site-directed mutagenesis protocol (Stratagene) with a 4 9 kb plasmid carrying the wildtype H3 gene (hH3) and 1 161 bp of 5'- flanking sequences (pSH12) as template
  • Primer pairs H3L9 and H3R9 were used to generate CTC and CGT codons in place ofthe AAG codon, respectively
  • the resulting plasmids (pSH12L9 and pSH12R9, respectively), and the wildtype control were linearized usingJft ⁇ l and cotransformed into N crassa strain N644 along with ///wdlll-hnea ⁇ zed pBT6 Approximately 1000 conidia from transformants grown en masse on solidified Vogel's sucrose medium
  • N. crassa has only a single copy ofthe hH3 gene (Woudt et al, Nucl Acids Res. 1 1 :5347- 5361 , 1983), but gene replacement by homologous recombination is inefficient in Neurospora.
  • replacement ofthe wild-type hH3 with the mutated versions might be lethal. It seemed possible, however, that the mutations would prove dominant or semi-dominant.
  • Advantage was taken ofthe methylated hph allele of N644 to test for loss of DNA methylation in random transformants generated with the mutated hH3 constructs.
  • Transformants generated with mutant or wildtype hH3 genes together with the cotransformation marker, Bml, were selected en masse on benomyl medium, then tested for expression of hph. About 500 asexual spores from each pool, representing ⁇ 30 bml R transformants, were spread on hygromycin plates (FIG 7B).
  • the hyg R transformants in each experiment contained a single ectopic copy ofthe mutant allele, which is unusual for Neurospora. Perhaps additional copies ofthe mutant hH3 genes were toxic, either directly or because they caused quelling, reducing H3 levels beyond the point that the cells could survive. Direct DNA sequencing of hH3 PCR products confirmed that the strains contained both the wildtype and mutant sequences (FIG 7C). These results strongly support the inference from other results reported herein that methylation of histone H3 is critical for DNA methylation.
  • Example 8 Generation of Heterochromatin by RIP
  • Constitutive heterochromatin is typically rich in moderately repeated sequences, such as transposons, and highly repeated sequences, such as satellite DNA, and displays a number of other identifying characteristics. It remains condensed after mitosis, replicates late in S-phase, shows low levels of genetic recombination, contains special forms of histones and, in organisms with DNA methylation, such as mammals and plants, it is hypermethylated (Hennig, Chromosoma 108:1-9, 1999).
  • heterochromatin protein HPI HPI
  • pombe depend on a similar set of silencing genes, including the chromo domain genes Sw ⁇ 6 and Clr4, which encode a HPI -like protein and a histone H3 MTase, respectively (Nakayama et al , Science 292 110- 1 13, 2001)
  • the chromo domain of HPI has recently been shown to recognize methylated Lys 9 of histone H3 (Bannister et al , Nature 410 120-124, 2001 , Lachner et al , Nature 410 116-120, 2001)
  • the finding reported herein that DNA methylation in Neurospora relies on histone methylation raises the possibility that sequences mutated by RIP, which constitute the bulk of methylated sequences of this organism and are found concentrated in centrome ⁇ c DNA (Cambare ⁇ et al , Mol Cell Biol 18 5465-5477, 1998), serve to nucleate heterochromatin RIP detects duplicated sequences, such as transposons (Margolin et al
  • Example 9 A New Paradigm: Histones as Signal Transducers for DNA Methylation The control of DNA methylation has remained enigmatic despite decades of intensive investigations in mammals, plants, and fungi
  • prokaryotic and eukaryotic DNA methyltransferases show striking structural similarities, prokaryotes offer an inappropriate paradigm for DNA methylation in eukaryotes
  • Bacterial DMTases require nothing more than DNA and a methyl-group donor for proper function and are sequence-specific
  • eukaryotic DMTases have substantial non-catalytic domains that reflect interactions with other proteins (Colot &
  • histones are subject to a variety of post-translational modifications (phosphorylation, methylation, acetylation, ubiquitination, and ADP-ribosylation) that can play informational roles in the cell (Strahl & Allis, Nature 403:41-45, 2000).
  • Acetylation currently the best understood modification, is controlled by histone acetylases (HATs) and histone deacetylases (HDACs), which typically act as transcriptional coactivators and corepressors, respectively.
  • HATs histone acetylases
  • HDACs histone deacetylases
  • TSA treatment or mutation of HDAC genes causes mis-localization of Swi-6 and other defects characteristic of disruption ofthe Clr4 HMTase (Nakayama et al, Science 292: 1 10-113, 2001 ; Grewal et al, Genetics 150:563-576, 1998; Ekwall et al, Cell 91 : 1021-1032, 1997). This is perhaps because methylation of K9 of histone H3 is inhibited by acetylation of lysine 9 or 14 (Rea et al, Nature 406:593-599, 2000; Nakayama et al, Science 292:1 10-1 13, 2001). Phosphorylation of Ser 10 also strongly inhibits methylation of lysine 9 (Rea et al, Nature 406:593-599, 2000), providing another illustration of how histones can integrate information from multiple inputs and act as signal transducers.
  • a defining feature of epigenetic states is that they promote their own propagation. Thus active chromosomal regions are rarely silenced and silenced regions are rarely activated. Holliday and Pugh and Riggs recognized that the symmetry of methylated sites (5'-CpG/GpC-5') in mammalian DNA would support a simple mechanism to propagate methylation patterns; all that was required was a DMTase specific for hemimethylated sites (Bestor & Tycko, Nat Genet 12:363-367, 1996). The "maintenance methylase" model was supported by evidence that methylation states are indeed propagated and by the discovery of DMTases that prefer hemimethylated substrates.
  • the classic maintenance model does not account for some observations, such as heterogeneous methylation in cell clones, spreading of methylation and stable propagation of methylation at non-symmetrical sites, as observed in Neurospora and other eukaryotes (Singer et al. , Mol. Cell. Biol. 15:5586-5597, 1995; Miao et al, J. Mol. Biol. 300:249-273, 2000; Selker et al, Science 262:1724-1728, 1993). With the demonstration herein that histone modifications can impact both de novo and maintenance DNA methylation, it is believed feasible that propagation of DNA methylation patterns in eukaryotes depends on feedback loops between modifications of chromatin proteins and DNA.
  • Histones H3 and H4 remain tightly bound to DNA in vivo, unlike histones H2A and H2B (Kimura et al. , J Cell Biol 153 : 1341 - 1354, 2001 ), consistent with the idea that these histones are involved in the propagation of epigenetic states.
  • the chromo domain is absent from the DIM-5 HMTase, and from the recently described G9a HMTase (Tachibana et al, J Biol Chem 276:25309-25317, 2001). Perhaps DNA methylation and associated factors (e.g., DMTases and methyl-DNA binding proteins) substitute for this potential self-reinforcing system. DMTases containing a chromo domain have been identified in plants (Lindroth et al, Science 292:2077-2080, 2001), suggesting that some DMTases may take cues directly from histones. A search of public databases with DIM-5 revealed a number of potential HMTases that may be involved in DNA methylation; certain of these potential HMTases are listed in Table 2.
  • Table 2 Examples of putative and/or known histone methyltransferases that may be involved in DNA methylation
  • Histones are subject to extensive posttranslational modifications including acetylation, phosphorylation, and methylation, primarily on their N-terminal tails that protrude from the nucleosome. Evidence accumulated over the past few years suggests that such modifications constitute a "histone code" that directs a variety of processes involving chromatin (Jenuwein and Allis, Science 293: 1074-1080, 2001 ; Strahl and Allis, Nature 403:41-45, 2000). Histone methylation represents the most recently recognized component ofthe histone code Most histone methylation occurs on lysine, though arginine methylation also occurs on histones H3 and H4 (Ma et al .
  • Lysine methylation is highly selective, with the best- characterized sites being K4 and K9 of histone H3
  • K9 methylation is associated with transc ⁇ ptionally inactive heterochromatin
  • K4 methylation is associated with transc ⁇ ptionally active euchromatin (Boggs et al , Nat Genet 30 73-76, 2002, Lift et al , Science 293 2453-2455, 2001 , Nakayama et al , Science 292 110- 113, 2001 , Nishioka et al , Gene Dev 16 479 ⁇ 189, 2002a)
  • K9 methylation has been implicated in transc ⁇ ptional silencing of Vietnamese genes such as those involved in cell cycle control (Nielsen et al ,
  • Drosophila genes involved in epigenetic processes Su(var)3-9, £n(zeste), and 7 ⁇ thorax (Jenuwein et al , Cell Mol Life Sci 54 80-93, 1998) Mammalian homologs of Drosophila SU(var)3-9 were shown to specifically methylate H3 at lysine 9 (Rea et al , Nature 406 593-599, 2000) Soon thereafter, related nstone lysine (K) ethy ransferases (HKMTs) in various species (see brief description of FIG 8) were found to methylate K4, K9, K27, or K36 of H3 methylated by a protein containing no SET domain (Feng et al , Curr Biol 12 1052-1058, 2002, Lacoste et al , J Biol Chem 277 30421-30424, 2002, Ng et al , Genes Dev 16 1518-1527, 2002, van Leeuwen et al
  • the approximately 130 amino acid SET domain is found in a large number of eukaryotic proteins as well as a few bacterial proteins and is not limited to histone H3 lysine 9 methyltransferases (HKMTs) More than 60 SET domain genes have been identified in humans (Pfa database, available online at The Sanger Institute) nearly 40 are found in the genome of Arabidopsis thaliana (Baumbusch et al , Nucleic Acids Res 29 4319-4333, 2001), and about 10 each are found in Drosophila and the fungi Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Neurosopora crassa SET proteins can be grouped into families according to the sequences surrounding this distinctive domain (Baumbusch et al , Nucleic Acids Res 29 4319-4333, 2001 , Kouza ⁇ des, Curr Opm Genet Dev 12 198-209, 2002)
  • This example provides a description ofthe elucidation ofthe crystal structure of Neurospora DIM-5, a HKMT, determined at 1.98 A resolution, as well as results of biochemical characterization and site-directed mutagenesis of key residues.
  • This SET domain protein bears no structural similarity to previously characterized AdoMet-dependent methyltransferases but includes notable features such as a triangular Zn3Cys9 zinc cluster in the pre-SET domain and a AdoMet binding site in the SET domain essential for methyl transfer.
  • the structure suggests a mechanism for the methylation reaction and provides the structural basis for functional characterization ofthe HKMT family and the SET domain.
  • This example is adapted from Zhang et al, Cell 11 1 : 1 17-127, 2002, which is inco ⁇ orated herein by reference in its entirety.
  • ORF including amino acid residues 17-318
  • pGEX-5X-3/DIM-5 Tamaru and Selker, MtfM e 414:277-283, 2001
  • the proteins were purified using Glutathione-Sepharose 4B (Amersham- Pharmacia), UnoQ6 (Bio-Rad), and Superdex 75 columns (Amersham-Pharmacia).
  • the GST tag was cleaved by applying thrombin to fusion proteins bound to the Glutathione-Sepharose column, leaving five additional residues (GSHMG) in front of amino acid 17 of DIM-5.
  • All purification buffers contained 1 mM DTT and no EDTA.
  • the protein was stored in the Superdex 75 column buffer containing 20 mM glycine (pH 9.8), 150 mM NaCl, 1 mM DTT, and 5% glycerol.
  • Se-containing DIM-5 (with five methionines) was expressed in a methionine auxotroph strain (B834) grown in the presence of Se- methionine, and the protein was purified similarly to the native protein.
  • the activity was assayed in a 20 ⁇ l reaction containing 50 mM glycine (pH 9.8), 2 mM DTT, 40-80 ⁇ M unlabeled AdoMet (Sigma), 0.5 ⁇ Ci [methyl- 3 H] AdoMet (78 Ci/mmol, NEN NET155H), 0.25-0.5 ⁇ g of DIM-5 protein, and 2-5 ⁇ g histones (calf thymus histones Sigma H4524, Roche 223565, or recombinant chicken erythrocyte histones, a gift from Dr. V. Ramakrishnan).
  • Amino acid replacements of DIM-5 (SEQ ID NO: 3) to yield R155H, W161F, Y204F, R238H, N241Q, H242K, D282K, and Y283F were made using Quik-Change site-directed mutagenesis protocol (Stratagene) using pXC379 and primer pairs to generate CAC, TTC, TTC, CAC, CAG, AAA, AAC, and TTC codons in place of AGG, TGG, TAC, AGG, AAC, CAC, GAC, and TAT codons, respectively (see SEQ ID NO: 2).
  • Quik-Change site-directed mutagenesis protocol (Stratagene) using pXC379 and primer pairs to generate CAC, TTC, TTC, CAC, CAG, AAA, AAC, and TTC codons in place of AGG, TGG, TAC, AGG, AAC, CAC, GAC, and TAT codons, respectively (see
  • the DIM-5 mutant 3C to 3S in which all three invariant cysteines in the post-SET region are replaced by serines, was generated by PCR using a mutagenic 3' primer. All mutants were sequenced to verify the presence ofthe intended mutation and the absence of additional mutations. The only exception is the Y204F mutant, which carries an additional Asp substitution (A24D) in the N-terminal region that was not observed in the structure. Mutant proteins, along with wild-type, were purified from 100-200 ml of induced cultures. A disposable column containing 0.5 ml of Glutathione-Sepharose 4B (Amersham-Pharmacia) was used for each mutant.
  • the mutant proteins were separated from GST by on-column thrombin cleavage and then used for enzymatic assay (using calf thymus histones Sigma H4524 as substrate), Ado-Met binding by crosslinking analysis, and analytical gel filtration chromatography for native protein size determination.
  • Zinc Content Analysis One sample of untreated and two samples of EDTA -treated DIM-5 protein (about 2 ml of 2 mg/ml each) was analyzed for the presence of 20 elements on a Thermo Jarrell-Ash Enviro 36 ICAP analyzer at the Chemical Analysis Laboratory ofthe University of Georgia at Athens.
  • the precise concentration ofthe untreated DIM-5 protein was determined by amino acid analysis (averaging two independent measurements) performed at the Keck Facilities at Yale University.
  • the extinction coefficient (29,559 M ⁇ cm "1 ) derived from the amino acid analysis was used to estimate the protein concentration ofthe EDTA-treated samples. Crystallography
  • SOLVE determined the positions of five selenium atoms: two of them (SeMet 233 and 248) were confirmed by Zn-phased map, and three of them (SeMet 75, 85, and 303) served as markers in the primary sequence during tracing.
  • the resultant model was refined against the data collected at wavelength of 1.0332 A in the resolution range of 24.8-1.98 A, using the X-PLOR program suite (Br ⁇ nger, X-PLOR. A System for X-Ray Crystallography and NMR, 3.1 edn, New Haven, CT: Yale University, 1992).
  • N-terminal 8 residues (17-24) (these may not be present in the native DIM-5 protein as there is an in-frame splicing site immediately after these residues); residues 89-99 ofthe pre-SET domain (these are deleted in many ofthe SUV39 proteins) (see FIG 8); and the majority ofthe C-terminal 34 amino acids (the C terminus is also highly variable in length and sequence among SET proteins except for the three-Cys post-SET region).
  • the nonglycine and nonproline residues 86% are in most favored and 14% in additional allowed regions of a Ramachandran plot (Laskowski, J. Appl. Crystallogr. 26:283-291, 1993).
  • Recombinant DIM-5 protein (residues 17 to 318 of Protein Data Bank accession number AF419248) was used for crystallographic studies (see Experimental Procedures). Electron density maps were calculated using multiwavelength anomalous diffraction data from three intrinsic zinc ions (Table 3). A model of DIM-5 was built and refined to 1.98 A resolution with a crystallographic R factor of 0.205 and R free value of 0.258. The final model includes 1913 protein atoms (with mean B values of 26.9 A 2 ), 3 zinc ions, and 103 water molecules, with rms deviations of 0.008 A and 1.5 A from ideality for bond lengths and angles, respectively.
  • the structural determination on DIM-5 allowed a structure-guided sequence alignment of SET proteins to be performed (FIG 8) that includes human SUV39 family proteins, all verified active HKMTs reported so far, and three bacterial SET proteins.
  • the 318 residue DIM-5 protein is the smallest member ofthe SUV39 family. It contains four segments: (1) a weakly conserved aminoterminal region (light blue), (2) a pre-SET domain containing nine invariant cysteines, (3) the SET region containing signature motifs of NHXCXPN and DY, and (4) the post-SET region containing three invariant cysteines.
  • the nine Cys pre-SET region is unique to the SUV39 family, while the post-SET region is also present in many members of SETl and SET2 families (Kouzarides, Curr. Opin. Genet. Dev. 12: 198-209, 2002), and even in one bacterial SET protein from Xylella fastidiosa (FIG 8).
  • Two active human HKMTs contain neither pre- nor post-SET regions: SET7 (Wang et al, Mol. Cell 8: 1207-1217, 2001) (also called SET9 [Nishioka e/ al, Gene Dev. 16:479- 489, 2002]) methylates lysine 4 of histone H3 and SET8 (Fang et al, Curr. Biol. 12:1086-1099, 2002) (also called PR-SET7 [Nishioka et al, Mol. Cell 9: 1201-1213, 2002]) methylates lysine 20 of H4.
  • the pre-SET residues form a 9 Cys cage enclosing a triangular zinc cluster (FIG 9A).
  • the SET residues are folded into six ⁇ sheets surrounding the catalytic methyl transfer site, with a helical cap ( ⁇ F) above the ⁇ sheets.
  • the amino-terminal residues appear to be critical to the structural integrity ofthe molecule: the 38 residue segment extends through nearly the entire back ofthe molecule in the orientation shown (FIG 9A), providing an edge strand ( ⁇ l , ⁇ 2, or ⁇ 3) to three separate ⁇ sheets and a 1 turn helix ⁇ xA connecting to the pre-SET triangular zinc cage.
  • the overall dimensions ofthe molecule are 60 x 50 x 30 A.
  • the triangular zinc cluster and the cofactor binding site are approximately 38 A apart, located at opposite ends ofthe molecule along the longest dimension (FIG 9A). A cleft can be seen running across from the cofactor binding site to the zinc cluster (FIG 9B).
  • the Pre-SET Domain Forms a Triangular Zinc Cluster
  • the pre-SET domain contains nine invariant cysteine residues that are grouped into two segments of five and four cysteines separated by various numbers of amino acids (46 in DIM-5). These nine cysteines coordinate three zinc ions to form an equilateral triangular cluster (FIG 9C). Each zinc ion is coordinated by two unique cysteines (six total), and the remaining three cysteine residues (C66, C74, and C128) are each shared by two zinc atoms, thus serving as bridges to complete the tetrahedral coordination ofthe metal atoms.
  • the distance between zinc atoms is -3.9 A, and the Zn-S distance is -2.3 A.
  • a similar metal-thiolate cluster can be found in metallothioneins that are involved in zinc metabolism, zinc transfer, and apoptosis (reviewed in Vasak and Hasler, Curr. Opin. Chem. Biol. 4:177-183, 2000). Methallothioneins often have two metal clusters: a
  • the SET Domain Forms the Active Site
  • the SET domain resembles a square-sided ⁇ barrel topped by a helical cap ( F, ⁇ G, ⁇ H, and ⁇ l).
  • the side chains of these two highly conserved segments are involved in (1) hydrophobic structural packing (1240 of ⁇ J and L279 and F281 of ⁇ l7), (2) intramolecular side chain-main chain interactions (after a sha ⁇ turn at P246, the side chain of N247 interacts with the main chain carbonyl oxygen of E278 and the main chain amide nitrogen of T280), (3) AdoMet and active site formation (R238 and F239 of ⁇ J, N241 :E278 pair, H242:D282 pair, and Y283).
  • DIM-5 were noticed. Under the current laboratory conditions, the enzyme is most active at ⁇ 10°C and nearly inactive at 37°C (FIG 10A). DIM-5 is extremely sensitive to salt, e.g., 100 mM NaCl inhibited its activity about 95% (FIG 10B). The enzyme also has a high pH optimum. DIM-5 showed maximal activity at ⁇ pH 9.8 (FIG 10C), although it showed strongest crosslinking to AdoMet around pH 8 (FIG 10D). Neither HKMT activity nor AdoMet binding were observed below pH 6.0. Cofactor Binding Pocket
  • AdoMet As the methyl donor.
  • AdoMet or its reaction product AdoHcy
  • consensus MTases These MTases are built around a mixed seven-stranded ⁇ sheet, and they include more than 20 structurally characterized MTases acting on carbon, oxygen, or nitrogen atom in DNA, RNA, protein, or small molecule substrates (Cheng and Roberts, Nucleic Acids Res. 29:3784-3795, 2001).
  • DIM-5 does not share structural similarity to any of these AdoMet-dependent proteins and appears to use a completely different means of interaction with its cofactor.
  • a difference electron density is observed in an open pocket on one end ofthe DIM-5 molecule opposite from the triangular zinc cluster (FIG 9A and FIG 1 1 ).
  • This density was inte ⁇ reted as the cofactor product, AdoHcy, which was present during crystal growth (see Experimental Procedures). Although part ofthe AdoHcy can be fit into the density, it is difficult to fit the entire molecule, particularly because there is no recognizable density for the adenine ring of AdoHcy. This could potentially reflect flexibility ofthe cofactor bound to DIM-5.
  • the flexibility may also result from low pH during crystallization (pH 5.4-5.6), a condition in which no UV crosslinking of AdoMet to the protein was observed (FIG 10D). At low pH the adenine ring might not interact stably enough with DIM-5 to be crosslinked to the protein or observed in the structure.
  • the side chains of these two arginines are locked in place by other conserved residues: the guanidino group of R155 is parallel to the plane ofthe W161 indole ring and ion pairs with D35; and the guanidino group of R238 is surrounded by three aromatic rings, F43, F239, and Y204, and its two terminal nitrogen atoms (N ⁇ and N ⁇ 2) form hydrogen bonds to the main chain carbonyl oxygen atoms of G230 and E231, respectively (FIG 11).
  • the cleft along the surface emanating from the presumed cofactor binding site is the likely binding site for the substrate polypeptide (FIG 9B).
  • One side of this cleft is formed by strand ⁇ 10 (green in FIG 12A) — the outermost strand ofthe ⁇ sheet (3t 9f 11J, 101) — and the other side is formed by the loop after strand ⁇ l7, which is the beginning ofthe disordered carboxy-terminal residues (286-299).
  • heterochromatin protein HP 1 binds to a methylated histone H3 peptide by inserting it as an antiparallel ⁇ strand between two 2 HPI strands, forming a hybrid three-stranded ⁇ sheet (Jacobs and Khorasanizadeh, Science 295:2080-2083, 2002; Nielsen et al, Nature 416:103-107, 2002).
  • ⁇ lO one side of the DIM-5 cleft is a strand
  • we superimposed the HPI ⁇ strand Drosophila HPI residues 60-62) onto DIM-5 strand ⁇ lO (residue 205-207) (FIG 12B).
  • H3 peptide e.g., Q5-S10 as observed in HPI
  • DIM-5 cleft FIG. 12C
  • residues Y283-V284-N285 following strand ⁇ l 7 on the other side ofthe peptide FIG. 12B
  • An induced-fit mechanism is used in HPI , in which the amino- terminal tail ofthe free HPI adopts a ⁇ strand-like conformation upon interacting with the H3 peptide (Nielsen et al, Nature 416: 103-10 ', 2002).
  • binding ofthe H3 peptide may induce residues Y283-V284-N285 of DIM-5 and subsequent disordered residues to adopt a more stable ⁇ strand conformation that interacts with the peptide to form a hybrid sheet.
  • the most interesting result ofthe docking experiment is the placement ofthe target K9 immediately next to the presumed cofactor binding site (FIG 12C) with the target nitrogen atom occupying the position of a water molecule (site 2 in FIG 1 1).
  • water site is the likely active site of DIM-5, where the terminal amino group (NH 3 ) ofthe substrate lysine would form a hydrogen bond with main chain carbonyl oxygen atom of R238.
  • Side chains of N241, H242, Y283, and Y204 form an inner circle immediately around site 2 (FIG 1 1).
  • Residues E278, D282, and Y178 form an outer circle via interactions with the inner-circle residues: E278 interacts with N241 , D282 interacts with H242, and Yl 78 interacts with Y283 via a water molecule (site 4) (FIG 1 1).
  • site 4 water molecule
  • site 4 water molecule
  • no acidic residue is immediately present in the proposed active site of DIM-5.
  • the presumptive active site of DIM-5 is reminiscent ofthe consensus NPPY motif involved in the aminomethylation of adenine or cytosine in DNA (Blumentbal and Cheng, Nat. Struct. Biol. 8: 101-103, 2001; Goedecke et al, Nat. Struct. Biol. 8: 121-125, 2001 ; Gong) and ofthe glutamine in peptide release factor (Heurgue-Hamard et al. , EMBO J. 21 -.169-11%, 2002; Nakahigashi et al, Proc. Natl. Acad. Sci. USA 99:1473-1478, 2002).
  • the invariant N241 and Y283 of DIM-5 are superimposable onto the first and the last amino acids of NPPY in Taql DNA adenine MTase (FIG 12D).
  • FOG 12D Taql DNA adenine MTase
  • the amino group (NH2) that becomes methylated is positioned for an in-line attack on AdoMet by hydrogen bonding to the backbone carbonyl connecting the two inflexible prolines (Goedecke et at, Nat. Struct. Biol. 8:121-125, 2001).
  • the equivalent backbone carbonyl in DIM-5 is probably that of R238.
  • the C terminus, including the post-SET region, is mostly disordered in the crystal except for the segment between residues 299 and 308 (FIG 9A and 9B).
  • This 10 residue segment identified through M303 in selenomethionine-substituted DIM-5 protein (see Experimental Procedures), was stabilized in the interface between two crystal lographic-related molecules. We hypothesize that this segment (along with the adjacent disordered residues) will adopt a different structure upon binding to substrate.
  • the post-SET region contains three conserved cysteine residues that appear to be essential for HKMT activity in the SUV39 family. Changing all three cysteines to serines (3C-S) abolished DIM-5 activity (FIG 10E), as did a Cys to Tyr substitution at C 1279 in SETDB 1 (Schultz et al,
  • the crystal structure of a histone H3 lysine 9 MTase, DIM-5 from N. crassa was determined and mutational and biochemical studies were carried out to illuminate the mechanism of this enzyme.
  • the highly conserved residues ofthe pre-SET region form a triangular zinc cluster, Zn3Cys9, and residues in the SET domain are essential for the cofactor binding and methyl transfer.
  • the SET domain also has a cleft that is the likely binding site for the methylatable amino-terminal tail of histone H3.
  • the post-SET region may also contribute to cofactor binding and catalysis by forming another zinc binding site in conjunction with a conserved cysteine near the active site.
  • Histone H3 methyltransferases have been implicated in various epigenetic processes.
  • DIM-5 histone methyltransferase which is essential for DNA methylation in Neurospora, trimethylates H3 Lys9.
  • DNA methylation and heterochromatin formation are tightly associated in organisms that show both features (Lachner et al, Curr Opin Cell Biol 14:286-298, 2002), it remains possible that they are triggered by distinct signals.
  • lysine 9 methylation has different consequences depending on the presence or absence of other histone modifications.
  • Recombinant DIM-5 was first assayed for methyltransferase activity using S-adenosyl- [methyl- 3 H]-Z,-methionine and synthetic methylated or unmodified H3 peptides. Reaction products were fractionated by SDS-PAGE and assayed for inco ⁇ oration of methyl groups by fluorography (FIG. 14A). Consistent with the expectation that DIM-5 is a H3 lysine 9 methyltransferase, DIM-5 methylated the 1-15 unmodified peptide derived from N-terminus of H3 but not a similar peptide that was trimethylated at lysine 9.
  • DIM-5 mass spectroscopy was used to follow the kinetics of mono-, di- and trimethyl transfer to unmodified or dimethylated lysine 9 in H3 peptides.
  • DIM-5 produced all three methylation states in the early phase ofthe reaction (FIG. 14D and 14F).
  • Trimethyl-lysine 9 had already became the dominant species at 30 minutes, when there was still substantial amount of unmethylated H3 peptide, and continued to increase while the relative amount of all other species decreased.
  • Dimethyl-lysine 9 was the least represented species throughout the reaction (FIG. 14D).
  • the methylation reactions may be distributive but DIM-5 simply prefers mono-, and especially dimethylated, substrates. Based on these findings, it is believed that trimethyl-lysine 9 H3 is the main product of DIM-5 in vitro. An additional question is whether DIM-5 preferentially generates trimethylated H3 lysine 9 in vivo.
  • asexual spores (-2 x 10 6 spores/ml) ofthe Neurospora wild-type strain N 150 (740R23-IVA) were germinated for 4.5 hours in 50 ml of Vogel's minimal liquid medium supplemented with 1.5 % sucrose and histidine at 32 °C with shaking (200 ⁇ m).
  • Cells of fission yeast strain SPG1335 were grown to 10 7 cells/ml in 50 ml yeast extract adenine medium as described (Noma et ⁇ l., Science 293: 1150-1 155, 2001).
  • PCR reactions (25 ⁇ l) included 50 mM KC1, 10 mM Tris-HCl (pH 9.0), 0.1 % Triton X-100, 2.5 M MgCl 2 , 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dCTP, 0.2 mM dGTP, 2.5 ⁇ Ci [ ⁇ - 32 P] dCTP and 1.25 U Taq polymerase (Promega). PCR products were fractionated in 4 % polyacrylamide gels and band intensities were quantified using a STORM 860 Phosphorimager (Molecular Dynamics).
  • pombe strain SPG1355 (Nakayama et ⁇ l., Cell 101 :307-17, 2000) carries an endogenous ur ⁇ 4 gene with a small deletion (ur ⁇ 4DS/E) and an ectopic ur ⁇ 4* gene integrated into the heterochromatic cenl locus (cenl::ur ⁇ 4) (FIG 15C).
  • Cultures of S. pombe strain SPG 1355 and the N. cr ⁇ ss ⁇ wild-type strain N 150 were mixed, the chromatin was fixed with paraformaldehyde, and immunoprecipitated with each antibody.
  • duplex PCR was conducted with sets of primers (SEQ ID NOs: 22 and 23 for ⁇ , SEQ ID NOs: 24 and 25 for punt, SEQ ID NOs: 26 and 27 for pen, and SEQ ID NOs: 28 and 29 for hH4) to amplify a pair of methylated and unmethylated regions ( ⁇ and pen or punt and hH4) from the DNA extracted from either immunoprecipitated or total chromatin.
  • the cenl::ura4 and ura4DS/E regions of S. pombe (FIG 15C) were also amplified from the same DNA sample.
  • PCR products were then fractionated by SDS-PAGE, quantified signals using a Phosphorimager and normalized the data based on results with total DNA.
  • the two active, nonmethylated genes pen and hH4 were efficiently precipitated with the anti-dimethyl-lysine 4 H3 antibody, whereas the inactive, methylated regions ( ⁇ and punt) were not.
  • the anti- trimethyl-lysine 9 H3 antibody preferentially precipitated both cytosine-methylated chromosomal regions.
  • pombe chromatin mixed in as an internal control were as expected (FIG 16C) (Noma et al, Science 293: 1 150-1 155, 2001). Specifically, the anti-dimethyl-lysine 4 H3 antibody preferentially precipitated ura4DS/E relative to cenl::ura4 (7.4 ⁇ 2.6-fold), whereas the anti- dimethyl-lysine 9 H3 antibody preferentially precipitated cenl::ura4 relative to ura4DS/E (10.8 ⁇ 4.1 -fold). These results confirmed that the dimethyl-lysine 9 antibody could immunoprecipitate chromatin with the dimethyl-lysine 9 modification and suggested that the silent regions of Neurospora and fission yeast are differentially methylated on lysine 9.
  • the anti-dimethyl-lysine 9 antibody failed to precipitate detectable chromatin associated with any of the four regions, as before.
  • the anti-dimethyl-lysine 4 and anti-trimethyl-lysine 9 antibodies preferentially precipitated the active (pen and hH4) and inactive, methylated ( ⁇ and punt) regions, respectively, in the wild-type strain (FIG 17A and 17B).
  • the dim-5 mutation markedly reduced the signals obtained with the trimethyl-lysine 9 antibody in the methylated regions, but did not appear to reduce the weaker signal observed with the active genes and did not completely eliminate signals with the methylated regions.
  • DIM-5 is indeed responsible for most, if not all, ofthe H3 trimethylation at lysine 9 detected in the methylated regions but is not responsible for the lower signal observed with the non-methylated genes.
  • the residual signal most likely reflects cross-reaction ofthe antibody with another epitope, but may reflect a low level of trimethylation by another enzyme.
  • mutation of dim-2 did not reduce signals in any ofthe regions examined, consistent with the conclusion that the DIM-2 DNA methyltransferase acts downstream ofthe DIM-5 histone methyltransferase, i.e., the dim-2 gene is not required for DIM-5 activity.
  • lysine 4 Neither the dim-5 nor the dim-2 mutation affected methylation of lysine 4 detected with the anti-dimethyl-lysine 4 antibody (FIG 17).
  • Reverse correlations have been observed for methylation of lysine 4 and lysine 9 of H3 in both fission yeast (Noma et ⁇ l., Science 293: 1150-5, 2001) and mammals (Litt et ⁇ l., Science 293:2453-5, 2001 ; Nishioka et ⁇ l, Genes Dev 16:479-89, 2002); lysine 9 methylation is found preferentially in heterochromatin and lysine 4 methylation is found preferentially in euchromatin.
  • H3 lysine 9 methylation can inhibit a H3 lysine 4 methyltransferase (Wang et ⁇ l., Mol Cell 8: 1207-17, 2001) and that H3 lysine 4 methylation can inhibit a H3 lysine 9 methyltransferase (Nishioka et ⁇ l., Genes Dev 16:479-89, 2002).
  • DIM-5 activity is also strongly inhibited by H3 lysine 4 methylation.
  • This disclosure provides in certain embodiments a novel HMTase that specifically methylates the lysine 9 residue of histone H3, and nucleic acids encoding this enzyme.
  • the disclosure further provides methods of using these molecules to influence DNA methylation and/or gene expression in eukaryotes, methods of screening for compounds that interact with the provided HMTase, and more generally methods of screening for compounds that are useful in treating, ameliorating, curing, or preventing methylation-related diseases or conditions (e.g., neoplasia).
  • methylation-related diseases or conditions e.g., neoplasia

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Abstract

La présente invention concerne une nouvelle histone méthyltransférase et des molécules d'acide nucléique la codant, ainsi que des variantes desdites molécules. L'invention concerne également des méthodes d'utilisation desdites molécules pour influencer la méthylation de l'histone et/ou la méthylation de l'ADN, ainsi que des méthodes de criblage de composés qui influencent la méthylation de l'histone et/ou de l'ADN. Ladite invention concerne également divers matériels.
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WO2004065592A2 (fr) * 2003-01-22 2004-08-05 Medical Research Council Structure de cristal de proteine
CN100577800C (zh) * 2007-01-29 2010-01-06 中国科学院遗传与发育生物学研究所 一种组蛋白甲基化转移酶及其编码基因与应用
FR3128470A1 (fr) * 2021-10-22 2023-04-28 IFP Energies Nouvelles Souche de champignon hyperproductrice de proteines

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US20020039776A1 (en) * 2000-06-09 2002-04-04 Thomas Jenuwein Mammalian SUV39H2 proteins and isolated DNA molecules encoding them

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US20020039776A1 (en) * 2000-06-09 2002-04-04 Thomas Jenuwein Mammalian SUV39H2 proteins and isolated DNA molecules encoding them

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DATABASE BIOSIS [Online] LEVESQUE ET AL.: 'Cytokine regulation of inducible nitric oxide synthase (NOS2) and NOS2 inhibitor-induced apoptosis and death in chronic lymphocytic leukemia cells', XP002970726 Retrieved from STN Database accession no. 2001:311668 & BLOOD vol. 96, no. 11, PART 1, 16 November 2000, page 159A Abstract *

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WO2004065592A2 (fr) * 2003-01-22 2004-08-05 Medical Research Council Structure de cristal de proteine
WO2004065592A3 (fr) * 2003-01-22 2005-03-24 Medical Res Council Structure de cristal de proteine
CN100577800C (zh) * 2007-01-29 2010-01-06 中国科学院遗传与发育生物学研究所 一种组蛋白甲基化转移酶及其编码基因与应用
FR3128470A1 (fr) * 2021-10-22 2023-04-28 IFP Energies Nouvelles Souche de champignon hyperproductrice de proteines

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